An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae

Streak appropriate S. cerevisiae strain on YPD agar. Incubate culture at 30°C for 2-3 days.

Transfer an individual colony to 5 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm overnight.

Dilute overnight culture to an OD600 of 0.2 in 40 ml of YPD. Incubate culture at 30°C with shaking at 220 rpm.

When the 40 ml culture reaches an OD600 of 0.3-0.4, cool centrifuge to 4°C and prepare 50 ml conical tubes, 2.0 ml microcentrifuge tubes, and 1.5 ml microcentrifuge tubes.

Add 5 ml of 100% methanol to a 50 ml conical tube for each sample to be collected. Keep tubes on dry ice. Three tubes are needed for each 40 ml culture.

When the 40 ml culture reaches an OD600 of 0.4-0.5, split the culture into three 10 ml aliquots in 50 ml conical tubes and return them to the incubator.

Each 10 ml culture represents one of three treatments:

A	B	C
Treatment	IAA treatment	4tU treatment
A	DMSO	4tU
B	IAA	4tU
C	DMSO	DMSO

Equilibrate cultures at 30°C for 5 min with shaking at 220 rpm.

During equilibration (or just prior to splitting the culture), prepare a 0.2 M IAA solution in DMSO. IAA is freshly prepared. To prepare a 0.2 M IAA solution, add 220 mg of IAA to 6 ml of DMSO and vortex until dissolved. Keep the IAA solution at room temperature covered with foil.

CRITICAL STEP: Add 50 μl of 0.2 M IAA solution or DMSO to the appropriate 10 ml culture. Vigorously mix the culture and return it to the incubator. The final concentration of IAA is 1 mM.

Incubate cultures at 30°C for 25 min with shaking at 220 rpm.

During IAA treatment, prepare a 2 M 4tU solution in DMSO. 4tU is freshly prepared. To prepare a 2 M 4tU solution, add 260 mg of 4tU to 1 ml of DMSO and vortex until dissolved. Keep the 4tU solution at room temperature covered with foil.

CRITICAL STEP: Immediately after IAA treatment, add 25 μl of 2 M 4tU solution or DMSO to the appropriate 10 ml culture. 4tU will precipitate when added to the culture; immediately and vigorously mix the culture and return it to the incubator. It is essential that cultures are uniformly mixed to ensure 4tU is dissolved and homogeneously distributed . The final concentration of 4tU is 5 mM.

Incubate cultures at 30°C for 4 min with shaking at 220 rpm.

CRITICAL STEP: Immediately after 4tU treatment, decant each 10 ml culture directly into 5 ml of 100% methanol in a 50 ml conical tube on dry ice (prepared in Step 5). Loosely cap the slurry and gently mix by swirling to ensure homogenization. Keep the fixed sample on dry ice. The final concentration of methanol is approximately 33%.

Gently mix the fixed sample and transfer 250 μl to a 1.5 ml microcentrifuge tube. Store the 250 μl aliquot at 4°C for cell counting. Estimate cell number using OD600 or count cells using a Neubauer chamber.

Gently mix the fixed sample and transfer 1.5 ml to a 2.0 ml microcentrifuge tube for western blotting. Centrifuge the sample at max speed for 1 min to pellet cells, discard the supernatant, and freeze cell pellets at -20°C.

Centrifuge the remaining sample at 3,000 g and 4°C for 10 min to pellet cells.

Carefully decant the supernatant and briefly invert the 50 ml conical tube on a paper towel to remove residual supernatant.

Adjust samples to a uniform cell number in DNA/RNA Shield. Use the following formula to calculate the appropriate volume of buffer for each sample: volume of buffer = (final OD600 of sample/0.70)*400. Thoroughly resuspend cells by pipetting.

Ensure cells are thoroughly resuspended and transfer 400 μl of the cell suspension to a 1.5 ml microcentrifuge tube.

PAUSE POINT: Flash freeze samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.

OPTIONAL STEP: If experiments require spike-in, prepare 4tU-treated S. pombe cells as described in Steps 22-35. Streak appropriate S. pombe strain on YE agar. Incubate culture at 30°C for 3-4 days.

Transfer an individual colony to 5 ml of YE. Incubate culture at 30°C with shaking at 220 rpm overnight.

Dilute overnight culture to an OD600 of 0.2 in 250 ml of YE. Incubate culture at 30°C with shaking at 220 rpm.

When the 250 ml culture reaches an OD600 of 0.6, cool centrifuge to 4°C and prepare 50 ml conical tubes and 1.5 ml microcentrifuge tubes.

Add 125 ml of 100% methanol to a 1 l flask for each culture to be collected. Keep flask on dry ice.

When the culture reaches an OD600 of 0.8, prepare a 2 M 4tU solution in DMSO as described in Step 11.

CRITICAL STEP: When the culture reaches an OD600 of 1.0, add 625 μl of 2 M 4tU solution. 4tU will precipitate when added to the culture; immediately and vigorously mix the culture and return it to the incubator.

Incubate the culture at 30°C for 4 min.

CRITICAL STEP: Immediately after 4tU treatment, decant the 250 ml culture directly into 125 ml of 100% methanol in a 1 l flask on dry ice (prepared in Step 26). Gently mix the slurry by swirling to ensure homogenization. Keep the fixed sample on dry ice.

Distribute 30 ml aliquots of the culture slurry to 50 ml conical tubes and centrifuge at 3,000 g and 4°C for 10 min to pellet cells.

Carefully decant supernatant and briefly invert 50 ml conical tubes on a paper towel to remove residual supernatant.

Thoroughly resuspend the cells in each 50 ml conical tube in 540 μl of DNA/RNA Shield by pipetting and pool cell suspensions in a fresh 50 ml conical tube on ice. Samples are adjusted to the equivalent of an OD600 of 7.0.

Ensure cells are thoroughly resuspended and distribute 500 μl aliquots of the cell suspension to 1.5 ml microcentrifuge tubes on ice.

PAUSE POINT: Flash freeze the samples on dry ice or in liquid nitrogen and store at -80°C until proceeding.

Supplement the following buffers and reagents with DTT (100 mM DTT working stock):

A	B	C	D
Treatment	Reagent vol. per sample	DTT vol. per sample	Final DTT conc.
RNA Lysis Buffer	400 μl	0.8 μl	0.2 mM
RNA Wash Buffer	1.5 ml	1.5 μl	0.1 mM
RNA Prep Buffer	400 μl	0.4 μl	0.1 mM
100% ethanol	550 μl	0.55 μl	0.1 mM
Nuclease-free water	50 μl	0.5 μl	1 mM

Thaw samples stored in DNA/RNA Shield at -80°C at room temperature.

Combine cells suspended in DNA/RNA Shield with RNA Lysis Buffer using one of the following options:

OPTION A (without spike-in): Combine 400 μl of RNA Lysis Buffer and 400 μl of cells suspended in DNA/RNA Shield.

OPTION B (with spike-in): Combine 360 μl of RNA Lysis Buffer and 400 μl of cells suspended in DNA/RNA Shield, and combine 500 μl of RNA Lysis Buffer and 500 μl of S. pombe cells suspended in DNA/RNA Shield.

Transfer cells suspended in DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube using one of the following options:

OPTION A (without spike-in): Transfer 800 μl of cells suspended in 1:1 DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube.

OPTION B (with spike-in): Transfer 760 μl of S. cerevisiae cells suspended in DNA/RNA Shield:RNA Lysis Buffer and 40 μl of S. pombe cells suspended in DNA/RNA Shield:RNA Lysis Buffer to a ZR Bashingbead Lysis Tube.

Process samples in a Mini-Beadbeater-24 at 3800 rpm for 45 s and immediately incubate on ice for 2 min.

Repeat Step 40 once.

While samples are incubating on ice, prepare the following reaction mixture in a 1.5 ml microcentrifuge tube on ice:

DNase I reaction mixture

A	B	C
Reagent	1X reaction vol. (μl)	Master mix vol. (μl)
DNA Digestion Buffer	75
DNase I, reconstituted (1 u/μl)	5
Total	80

Centrifuge samples at 16,000 g for 1 min to pellet debris.

CRITICAL STEP: Transfer 550 μl of the cleared supernatant to a Zymo-spin IIICG Column in a collection tube and centrifuge at 16,000 g for 30 s. Save the flow through.

Add an equal volume (550 μl) of 100% ethanol to the flow through. Thoroughly mix samples by pipetting.

Transfer 700 μl of the mixture to a Zymo-spin IICR Column in a collection tube and centrifuge at 16,000 g for 30 s. Discard flow through.

Repeat Step 46 with the remaining mixture volume.

Add 400 μl of RNA Wash Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.

Gently mix DNase I reaction mixture (prepared in Step 42) by inversion and add 80 μl directly to the column matrix. Incubate the reaction at room temperature for 15 min.

Add 400 μl of RNA Prep Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.

Add 700 μl of RNA Wash Buffer to the column and centrifuge at 16,000 g for 30 s. Discard flow through.

Add 400 μl of RNA Wash Buffer to the column and centrifuge at 16,000 g for 1 min to ensure complete removal of buffer.

Carefully transfer the column to a labeled nuclease-free 1.5 ml microcentrifuge tube.

Add 50 μl of nuclease-free water prewarmed to 50°C directly to the column matrix, incubate at room temperature for 2 min, and centrifuge at 16,000 g for 30 s.

PAUSE POINT: Store purified RNA at -20°C (up to four weeks) or -80°C until proceeding.

Thaw RNA samples on ice. Quantify RNA using a Nanodrop.

Add 5 μg of total RNA to a 1.5 ml microcentrifuge tube on ice. Adjust the volume of each sample to 20 μl with nuclease-free water.

Prepare a 100 mM iodoacetamide solution in DMSO. Iodoacetamide is freshly prepared. To prepare a 100 mM iodoacetamide solution, add 9.25 mg of iodoacetamide to 500 μl of DMSO (1 mg of iodoacetamide to 54 μl of DMSO). Keep the iodoacetamide solution at room temperature covered with foil. Do not store unused iodoacetamide.

Prepare the following reaction mixture in a 1.5 ml microcentrifuge tube and mix well by pipetting:

Alkylation reaction mixture

A	B	C
Reagent	1X reaction vol. (μl)	Master mix vol. (μl)
100 mM iodoacetamide	5
0.5 M sodium phosphate buffer (pH 8.0)	5
DMSO	20
Total	30

Add 30 μl of reaction mixture to the 1.5 ml microcentrifuge tube containing 5 μg of total RNA in 20 μl nuclease-free water (prepared in Step 57). The final concentration of iodoacetamide is 10 mM.

Gently mix the reaction and incubate in a thermomixer at 900 rpm and 50°C for 15 min in the dark.

Add 1 μl of 1 M DTT to stop the reaction. Briefly vortex to mix. Exposure to light is acceptable following the addition of DTT.

Add 1 μl of glycogen (20 mg/ml) and 5 μl of 3 M sodium acetate (pH 5.2) to each sample. Briefly vortex to mix.

Add 125 μl of 100% ethanol to each sample. Briefly vortex to mix and incubate at -80°C for 30 min.

Centrifuge samples for 30 min at 21,000 g and 4°C to pellet precipitated RNA.

Carefully decant the supernatant. Take care not to dislodge pellet.

Add 1 ml of 80% ethanol to each sample. Centrifuge samples for 10 min at 21,000 g and 4°C to wash pellet.

Carefully decant the supernatant. Take care not to dislodge pellets. Carefully inspect samples and remove remaining ethanol with a 20 μl pipet.

Air dry samples for 5 min with lid open. Do not over dry samples.

Resuspend RNA in 30 μl of nuclease-free water.

PAUSE POINT: Store alkylated RNA at -20°C (up to four weeks) or -80°C until proceeding.

RNA fragmentation and cDNA synthesis.

Thaw alkylated RNA samples on ice. Quantify RNA using an RNA-specific fluorometry method (i.e., Qubit RNA BR or HS assay kit).

Heat samples to 42°C for 60 min followed by 70°C for 10 min in a thermocycler with heated lid. Cool samples to 37°C in a thermocycler. Proceed immediately to RNA removal (Step 73.1).

Add 200 ng of RNA to a 0.2 ml PCR tube or strip on ice. Adjust the volume of each sample to 5 μl with nuclease-free water.

Prepare the following reaction mixture in a 0.2 ml PCR tube (or 1.5 ml microcentrifuge tube) on ice and mix well by pipetting:

Fragmentation reaction mixture

A	B	C
Reagent	1X reaction vol. (μl)	Master mix vol. (μl)
5X reverse transcription buffer	2
20 mM dNTP mix	0.5
20 μM IRA_UMI_24dTVN oligo	0.5
Total	3

Prepare the following reaction mixture in a 0.2 ml PCR tube (or 1.5 ml microcentrifuge tube) on ice and mix well by pipetting:

Reverse transcription (RT) reaction mixture

A	B	C
Reagent	1X reaction vol. (μl)	Master mix vol. (μl)
20 mM DTT	1
20 μM IFA-isoTSO oligo	0.5
Reverse transcriptase (200 u/μl)	0.5
Total	2

Bring the RNA and fragmentation reaction mixture to room temperature for 2-5 minutes and add 3 μl of the fragmentation reaction mixture to each sample. Mix well by pipetting.

CRITICAL STEP: Incubate samples at 94°C for exactly 4 min in a thermocycler with heated lid to fragment the RNA. Cool samples to 42°C in a thermocycler. Fragmentation time can be optimized to generate libraries of different insert sizes.

During RNA fragmentation, bring the RT reaction mixture (prepared in Step 72.4) to room temperature for 2-5 minutes.

Remove samples from the thermocycler, briefly centrifuge, and add 2 μl of the RT reaction mixture to each sample. Mix well by pipetting.

Briefly centrifuge and return samples to a thermocycler at 42°C.

RNA removal and single-stranded DNA purification.

Add 1 μl of RNase H to each sample and mix well by pipetting. Centrifuge briefly at room temperature to collect samples.

Heat samples to 37°C for 15 min in a thermocycler with heated lid. Proceed directly to speedbead purification.

Purify the single-stranded DNA (ssDNA). Add 39 μl of DNA buffer and 1 volume (50 μl) of speedbeads to each sample and proceed as described in the general method for speedbead purification (Step 76).

Elute DNA in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C.

Transfer 20 μl of the cleared supernatant to a fresh PCR tube.

PAUSE POINT: Store purified DNA at -20°C (up to eight weeks) or -80°C until proceeding.

Barcoding and enrichment PCR and library purification.

Transfer 10 μl of purified DNA to a 0.2 ml PCR tube on ice.

Add 0.8 volumes (24 μl) of DNA binding buffer to the 0.2 ml PCR tube containing the library and bead suspension. Proceed as described in the general method for speedbead purification (Step 76).

Elute the library in 21 μl of 10 mM Tris-HCl (pH 8.0) prewarmed to 50°C.

Transfer 20 μl of the cleared supernatant to a fresh PCR tube.

PAUSE POINT: Store purified libraries at -20°C (up to four weeks) or -80°C until proceeding.

Add 2.5 μl of an appropriate 5 μM i5/i7 primer mix to each sample on ice.

Add 12.5 μl of 2X Ex Premier master mix to each sample on ice. Mix thoroughly by pipetting.

Barcode and enrich the library using PCR. Perform initial denaturation at 98°C for 45 s, followed by 12-18 cycles of amplification (98°C for 10 s, 60°C for 15 s, and 68°C for 15 s), and bring the reactions to 20°C.

Purify the library. Add 25 μl of DNA buffer and 0.8 volumes (40 μl) of speedbeads to the 0.2 ml PCR tube containing the amplified library.

Mix well by pipetting up and down a minimum of 10 times. Alternatively, vortex samples on setting 4 for 3-5 seconds. If samples require centrifugation after mixing, stop the centrifuge before beads settle.

Incubate samples at room temperature for 5 min.

Place samples on magnetic stand for 2-5 min (or until solution is completely clear) to collect beads. Carefully remove and discard cleared supernatant without disturbing the bead pellet.

Remove samples from the magnetic stand. Add 30 µl of DNA buffer and mix well by pipetting or vortexing. Ensure beads are fully resuspend and incubate samples at room temperature for 2 min.

Library quality control, quantification, pooling, and sequencing.

Assess library quality using an Agilent Bioanalyzer or agarose gel electrophoresis.

Quantify libraries using a DNA-specific fluorometry method (i.e., Qubit dsDNA BR or HS assay kit).

Pool equimolar concentrations (i.e., 5 nM) of each library. The recommended final concentration of the pool is 5 nM.

Sequence the library pool on an Illumina instrument (e.g., NovaSeq 6000). The recommended sequencing depth is 10 million reads per sample.

Equilibrate speedbeads to room temperature and mix thoroughly.

CRITICAL STEP: Carefully add the indicated amount of speedbeads to the sample.

Mix well by pipetting up and down a minimum of 10 times. Alternatively, vortex samples on setting 4 for 3-5 seconds. If samples require centrifugation after mixing, stop the centrifuge before beads settle.

Incubate samples at room temperature for 5 min. Prolonged incubation or incubation at low temperature will increase binding of small nucleic acids (e.g., adapter dimers).

Place samples on magnetic stand for 2-5 min (or until solution is completely clear) to collect beads. Keep samples on magnetic stand for Steps 81-85.

Carefully remove and discard cleared supernatant without disturbing the bead pellet.

Add 200 μl of 80% ethanol (freshly prepared with MilliQ water) and incubate for 30 s. Carefully remove ethanol and discard without disturbing the bead pellet.

Repeat Step 82 once. Carefully inspect samples and remove remaining ethanol using a 10 μl pipet.

CRITICAL STEP: Air dry samples for 2 min with lid open. Do not over-dry samples. Speedbeads (and other SPRI products) will clump and can not be fully resuspended during elution if samples are over-dried.

Remove samples from the magnetic stand. Add the indicated volume of appropriate solution for elution and mix well by pipetting or vortexing (see Step 78).

Incubate mixture at room temperature for 2-5 min.

Place samples on a magnetic stand for 2 min (or until solution is completely clear) to collect beads.

Transfer cleared supernatant (volume of solution added for elution less 1 μl) to a fresh PCR tube.

Download and install the following software and dependencies:

• fastp (version 0.23.2)
• BBMAP (version 39.06)
• Python (version 3.10.2)
• SLAM-DUNK (version 0.4.3)
• NextGenMap (version 0.5.5)
• SAMtools (version 1.18)
• VarScan 2 (version 2.4.5)
• R (version 4.1.2-mkl)
• Fastqc (version 0.12.1)
• Multiqc (version 1.21)

Download reference genome and annotation files. The reference genome assembly and annotation for S. cerevisiae strain S288C (version R64-3-1, RefSeq Assembly GCF_000146045.2) can be retrieved from the NCBI Datasets repository.

Download the reference BED file attached below. This BED file will be used to define counting windows in SLAM-DUNK.

CRITICAL STEP: Set required variables using the commands below. Directories or files within quotations must be changed appropriately before running.

DATA="path_to_zipped_fastq_files"
SAMPLES="file_listing_fastq_files_to_process"
DIRECTORY="path_to_outfiles"
BBMAP_RESOURCES="path_to_bbmap_reference_files"
GENOME_scer="reference_S.cerevisiae_genome_file"
BED_scer="reference_S.cerevisiae_bed_file"

Make subdirectories to store outfiles created in Steps 94-101 using the following commands:

mkdir -p $DIRECTORY/logs/fastqc/fastq
mkdir -p $DIRECTORY/logs/multiqc/fastq
mkdir -p $DIRECTORY/fastp
mkdir -p $DIRECTORY/logs/fastp
mkdir -p $DIRECTORY/bbduk
mkdir -p $DIRECTORY/logs/bbduk
mkdir -p $DIRECTORY/logs/fastqc/bbduk
mkdir -p $DIRECTORY/logs/multiqc/bbduk
mkdir -p $DIRECTORY/slamdunk_scer
mkdir -p $DIRECTORY/slamdunk_scer/count_twotcreadcount
mkdir -p $DIRECTORY/slamdunk_scer/alleyoop
mkdir -p $DIRECTORY/logs/multiqc/slamdunk_scer

Run fastqc to assess raw data quality and multiqc to compile the fastqc output using following the commands:

echo "running fastqc on raw data for sample:"
for sample_name in $(cat $SAMPLES | sed "s/_001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(fastqc \
		$DATA/${sample_name}_001.fastq.gz \
		--outdir $DIRECTORY/logs/fastqc/fastq \
		--threads 64 \
		--quiet \
		&> /dev/null)

done


echo "running multiqc on raw data..."
multiqc	\
	$DIRECTORY/logs/fastqc/fastq \
	--outdir $DIRECTORY/logs/multiqc/fastq \
	--quiet \
	&> /dev/null

Run fastp to extract unique molecular identifiers (UMIs) using the following commands:

echo "processing UMIs for sample:"
for sample_name in $(cat $SAMPLES | sed "s/_R._001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(fastp \
		--in1 $DATA/${sample_name}_R1_001.fastq.gz \
		--out1 $DIRECTORY/fastp/${sample_name}_umi_R1_001.fastq.gz \
		--in2 $DATA/${sample_name}_R2_001.fastq.gz \
		--out2 $DIRECTORY/fastp/${sample_name}_umi_R2_001.fastq.gz \
		--disable_adapter_trimming \
		--disable_quality_filtering \
		--disable_length_filtering \
		--umi \
		--umi_loc read2 \
		--umi_len 12 \
		--json $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.json \
		--html $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.html \
		2> $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.log)

done

Run bbduk from the BBMap package to trim adapter sequences and polyA tails using the following commands:

echo "trimming adapters for sample:"
for sample_name in $(ls -1 $DIRECTORY/fastp | sed "s/_umi_R._001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(bbduk.sh \
		in=$DIRECTORY/fastp/${sample_name}_umi_R1_001.fastq.gz \
		out=$DIRECTORY/bbduk/${sample_name}_trimmed_R1_001.fastq.gz \
		ref=$BBMAP_RESOURCES/truseq.fa.gz,$BBMAP_RESOURCES/polyA.fa.gz \
		k=13 \
		ktrim=r \
		mink=5 \
		qtrim=r \
		trimq=10 \
		minlength=20 \
		threads=16 \
		2> $DIRECTORY/logs/bbduk/${sample_name}_trimmed_bbduk.log)

done

Run fastqc to assess preprocessed data quality and multiqc to compile the fastqc output using following the commands:

echo "running fastqc on preprocessed data..."
fastqc \
	$DIRECTORY/bbduk/*.fastq.gz \
	--outdir $DIRECTORY/logs/fastqc/bbduk \
	--threads 64 \
	--quiet \
	&> /dev/null


echo "running multiqc on preprocessed data..."
multiqc	\
	$DIRECTORY/logs/fastqc/bbduk \
	--outdir $DIRECTORY/logs/multiqc/bbduk \
	--quiet \
	&> /dev/null

Run slamdunk all from the SLAM-DUNK package to align data and quantify total reads and reads that have ≥1 T>C conversions using the following commands:

echo "running slamdunk all..."
slamdunk all \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--outputDir $DIRECTORY/slamdunk_scer \
	--trim-5p 5 \
	--topn 100 \
	--multimap \
	--min-coverage 10 \
	--var-fraction 0.8 \
	--max-read-length 151 \
	--min-base-qual 26 \
	--skip-sam \
	--threads 24 \
	$DIRECTORY/bbduk/*.fastq.gz

Rename the count directory created by slamdunk all using the following command:

mv $DIRECTORY/slamdunk_scer/count $DIRECTORY/slamdunk_scer/count_onetcreadcount

Run slamdunk count from the SLAM-DUNK package to quantify reads that have ≥2 T>C conversions using the following commands:

echo "running slamdunk count..."
slamdunk count \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--outputDir $DIRECTORY/slamdunk_scer/count_twotcreadcount \
	--max-read-length 151 \
	--min-base-qual 26 \
	--conversion-threshold 2 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam

Run the following alleyoop commands from the SLAM-DUNK package to summarize and merge slamdunk output:

echo "running alleyoop..."
alleyoop rates \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/rates \
	--reference $GENOME_scer \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop tccontext \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tccontext \
	--reference $GENOME_scer \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop utrrates \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/utrrates \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--multiTCStringency \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop snpeval \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/snpeval \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--min-coverage 10 \
	--var-fraction 0.8 \
	--multiTCStringency \
	--max-read-length 151 \
	--min-base-qual 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop summary \
	--output $DIRECTORY/slamdunk_scer/alleyoop/summary_onetcreadcount.tsv \
	--tcountDir $DIRECTORY/slamdunk_scer/count_onetcreadcount \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop summary \
	--output $DIRECTORY/slamdunk_scer/alleyoop/summary_twotcreadcount.tsv \
	--tcountDir $DIRECTORY/slamdunk_scer/count_twotcreadcount \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_totalreadcount.tsv \
	--column "ReadCount" \
	$DIRECTORY/slamdunk_scer/count_onetcreadcount/*.tsv


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_onetcreadcount.tsv \
	--column "TcReadCount" \
	$DIRECTORY/slamdunk_scer/count_onetcreadcount/*.tsv


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_twotcreadcount.tsv \
	--column "TcReadCount" \
	$DIRECTORY/slamdunk_scer/count_twotcreadcount/*.tsv


alleyoop tcperreadpos \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tcperreadpos \
	--reference $GENOME_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop tcperutrpos \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tcperutrpos \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop dump \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/dump \
	--reference $GENOME_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam

Run multiqc to compile output from the SLAM-DUNK package using the following commands:

echo "running multiqc on slamdunk output..."
multiqc\
	$DIRECTORY/slamdunk_scer/alleyoop \
	--outdir $DIRECTORY/logs/multiqc/slamdunk_scer \
	--quiet \
	&> /dev/null

Key results from this analysis are the three count tables described below. These files are located in the alleyoop directory (i.e., $DIRECTORY/slamdunk_scer/alleyoop).

• merge_totalreadcount.tsv - a table (tab-separated values) providing total read counts for all processed samples
• merge_onetcreadcount.tsv - a table (tab-separated values) providing read counts with ≥1 T>C conversions for all processed samples
• merge_twotcreadcount.tsv - a table (tab-separated values) providing read counts with ≥2 T>C conversions for all processed samples

Additional processing, analysis, and plotting can be performed in R (version 4.2.3) (https://www.R-project.org/) and RStudio (version 2023.12.0+369) (http://www.rstudio.com/) based on specific experimental goals.

OPTIONAL STEP: If experiments used S. pombe spike-in cells, set variables and make subdirectories using the commands below. Download the reference S. pombe genome assembly. Download the reference S. pombe BED file attached below. Repeat Steps 98-103 to generate spike-in count tables.

GENOME_spom="reference_S.pombe_genome_file"
BED_spom="reference_S.pombe_bed_file"

mkdir -p $DIRECTORY/slamdunk_spom
mkdir -p $DIRECTORY/slamdunk_spom/count_conversionthreshold
mkdir -p $DIRECTORY/slamdunk_spom/alleyoop
mkdir -p $DIRECTORY/logs/multiqc/slamdunk_spom