Barcoded Calling Cards and Transcriptomes: Library Preparation

Harvest cells. Do not overload columns. If you have more than 10⁷ cells, split cells in half and process on two columns, then merge the RNA pools.

Add TRI Reagent directly to cells in a dish. Use the following table as a guide.

A	B
# cells	Add TRI Reagent
< 1e6	300 µl
< 5e6	600 µl

Lyse the cells and transfer into 1.7 mL tube.

Add an equal volume ethanol (95-100%). Homogenize the lysate by vortexing briefly.

Transfer 700 ul of this mixture into a Zymo-Spin IICR Column spin column placed in a 2 ml collection tube. Centrifuge for 30 seconds at ≥ 8,000g. Ensure no liquid remains on the column membrane. Repeat centrifugation for samples > 700 µl.

Add 400 µL RNA PreWash buffer to column and centrifuge. Discard the flow-through.

Add 700 µl RNA Wash Buffer to the column and centrifuge briefly. Discard flow-through and centrifuge for 2 minutes. Transfer column into an RNase-free tube.

Elute RNA by adding 50 µl DNase/RNase-free water or te buffer to column and centrifuging.

Measure RNA concentration using Nanodrop or Qubit.

For first strand synthesis by reverse transcription, process samples according to experimental design. Use a unique BRB-seq primer for each transcriptome to be captured.

Prepare the reverse transcription (RT) reaction mix:

• 2 µg total RNA
• 2 µl of 25 µM BRB-seq_dT30VN primer (e.g., pSeq1-BC1-UMI-dtVN)
• 1 µl of 10 mM dNTPs
• Raise to 14 µl with ddH₂O

Incubate RT mix at 65°Cfor 0h 5m 0s

Place on ice for 1 minute.

Create 1x Maxima RT buffer:

• For 5 or fewer samples, combine 1 µL of 5X Maxima RT buffer with 4 µL of ddH₂O.
• Mix by pipetting and store on ice.

Create a 0.5x Maxima RT H Minus enzyme dilution:

• Mix an equal volume of Maxima RT H Minus Enzyme with the 1x Maxima RT buffer made in previous step (e.g. 2 µL of Enzyme + 2 µL of 1x buffer).

You will need 1 µL of the 0.5x enzyme dilution for every sample being processed. Avoid pipetting volumes < 1 µL.

Add the following to the RT mix:

• 4 µl 5X Maxima RT Buffer
• 1 µl RNasin RNase Inhibitor
• 1 µl of 0.5X Maxima RT H Minus enzyme (1:1 mixture of 1X Maxima RT Buffer and Maxima RT H Minus enzyme = 100 U)
• 1 uL SMART_TSO (25 µM)

Mix by pipetteing and incubate at 50°Cfor 1h 0m 0s

Heat inactivate the reaction by incubating at 85°Cfor 0h 10m 0s

Column purify using NucleoSpin Gel and PCR Clean-up kit to remove carry-over oligoDT primers.

• If pooling, combine 3-5 µl of each sample before column purification.

   Bring total volume of pooled or individual samples to `100µL` by adding water.  
  
  
  
   Add 200 µl NT1 and proceed with column purification according to manufacturer's instructions.
  
  
  
   Elute in 30-50 µl DNAse/RNAse free water. 
  

cDNA can be stored at –20 ºC for long term, or 4 ºC until downstream processing

This PCR will specifically amplify self-reporting transcripts from cDNA libraries. Prepare the following solution:

• 25 µl 2X Kapa HiFi HotStart ReadyMix

• 1 µl of 25 µM Reverse Primer (partial seq1)

• 4 µl of purified cDNA*

• 19 µl of ddH₂O

• 1 µl of Forward Primer, either: 25 µM SRT_PAC_F1 primer, if using PB-SRT-Puro

    25 µM SRT_tdTomato_F1, if using PB-SRT-tdTomato
  

Perform PCR using the following thermocycling parameters:

• 95ºC for 3 minutes
• 20 cycles of:
• 98ºC for 20 seconds
• 65ºC for 30 seconds
• 72ºC for 5 minutes
• 72ºC for 10 minutes
• 4ºC forever

If desired, this PCR will amplify full-length transcriptomes from cDNA libraries. Prepare the following solution:

• 25 µl 2X Kapa HiFi HotStart ReadyMix
• 1 µl of 25 µM Reverse Primer (partial seq1)
• 1 ul of 25 uM Forward Primer (SMART)
• 4 µl of pooled and purified cDNA
• 19 µl of ddH₂O

Perform PCR using the following thermocycling parameters:

• 95ºC for 3 minutes
• 10 cycles of:
• 98ºC for 20 seconds
• 60ºC for 30 seconds
• 72ºC for 6 minutes
• 72ºC for 10 minutes
• 4ºC forever

Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.

Add 30 µl beads to each 50 µl PCR mixture (0.6x ratio). Mix by pipetting 10 times until evenly dispersed.

Incubate at room temperature for 0h 5m 0s

Place on a magnetic rack for 2 minutes. Aspirate supernatant.

Add 200 µl of freshly-prepared 70% ethanol off the magnetic rack and mix. Place on magnetic rack and incubate ≥ 30 seconds. Aspirate ethanol.

Repeat Step 24 on the magnetic rack.

Air dry the beads at room temperature for 2 minutes. Do not over dry.

Remove the tube from the magnetic rack. Add 20 µl ddH₂O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes.

Place on magnetic rack for 1 minute, or until supernatant is clear.

Transfer supernatant to new tube. Quantify product on Tapestation D5000.

The tagmentation protocol fragments the long PCR products into libraries suitable for sequencing.

Preheat thermocycler to 55°C

Take 1 ng of PCR product and resuspend in a total of 5 µl ddH₂O in a PCR strip tube.

Add 10 µl of Nextera Tagment DNA (TD) Buffer and 5 µl of Amplicon Tagment Mix (ATM). Pipette to mix and briefly spin down; bubbles are normal. Incubate at 55°Cfor 0h 5m 0s

Add 5 µl of Neutralization Tagment (NT) Buffer. Pipette to mix and briefly spin down; bubbles are normal. Incubate at room temperature for 0h 5m 0s

For SRT libraries, add the following to each PCR tube in order:

• 15 µl Nextera PCR Mix (NPM)
• 8 µl ddH₂O
• 1 µl of 10 µM barcoded piggyBac primer (e.g., P5_BC_SRT_STAGGER1)
• 1 µl of 10 µM indexed Nextera N7 primer (e.g. Nextera_N701)

Perform PCR using the following thermocycling parameters:

• 72ºC for 3 minutes
• 95ºC for 30 seconds
• 16 cycles of:
• 95ºC for 10 seconds
• 52ºC for 30 seconds
• 72ºC for 30 seconds
• 72ºC for 5 minutes
• 4ºC forever

In parallel with Calling Card libraries, prepare BRB-seq libaries with tagmentation.

Preheat thermocycler to 55°C

Take 1 ng of PCR product and resuspend in a total of 5 µl ddH₂O in a PCR strip tube.

Add 10 µl of Nextera Tagment DNA (TD) Buffer and 5 µl of Amplicon Tagment Mix (ATM). Pipette to mix and briefly spin down; bubbles are normal. Incubate at 55°Cfor 0h 5m 0s

Add 5 µl of Neutralization Tagment (NT) Buffer. Pipette to mix and briefly spin down; bubbles are normal. Incubate at room temperature for 0h 5m 0s

For BRB-seq library amplification, add the following to each PCR tube in order:

• 15 µl Nextera PCR Mix (NPM)
• 8 µl ddH₂O
• 1 µl of 10 µM P5-index-seq1 primer
• 1 µl of 10 µM indexed Nextera N7 primer (e.g. Nextera_N701)

Perform PCR using the following thermocycling parameters:

• 72ºC for 3 minutes
• 95ºC for 30 seconds
• 16 cycles of:
• 95ºC for 10 seconds
• 55ºC for 30 seconds
• 72ºC for 30 seconds
• 72ºC for 5 minutes
• 4ºC forever

Purify PCR libraries using AMPure XP beads. Vortex AMPure XP beads to resuspend them. Beads should be brought to room temperature for at least 30 minutes prior to use.

Add 35 µl beads to each 50 µl PCR mixture (0.7x ratio). Mix by pipetting 10 times until evenly dispersed.

Incubate at room temperature for 0h 5m 0s

Place on a magnetic rack for 2 minutes. Aspirate supernatant.

Add 200 µl of freshly-prepared 70% ethanol off the magnetic rack and mix. Place on magnetic rack and incubate ≥ 30 seconds. Aspirate ethanol.

Repeat Step #46 on the magnetic rack.

Air dry the pellet at room temperature for 2 minutes.

Remove the tube from the magnetic rack. Add 11 µl ddH₂O to elute PCR products. Mix by pipetting until evenly dispersed. Incubate off the rack for 2 minutes.

Place on magnetic rack for 1 minute, or until supernatant is clear. Transfer supernatant to new tube.

Measure library concentration on a TapeStation device with a High Sensitivity D1000 ScreenTape. Libraries should be smoothly distributed between 300-600 bp.

Libraries can be sequenced on any Illumina sequencing platform.