METATAC V.1

Prepare METATAC Reagents

ATAC resuspension buffer (ATAC-RSB) 5mL ATAC resuspension buffer (ATAC-RSB)

Mix 50µL 1 M Tris-HCl pH 7.5, 10µL 5 M NaCl, 15µL 1 M MgCl2, and bring the final volume to 5mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

A	B	C
Reagents	5 mL	Final conc.
1 M Tris-HCl pH 7.5	50 µL	10 mM
5 M NaCl	10 µL	10 mM
1 M MgCl2	15 µL	3 mM
Nuclease-free H2O	4925 µL
Total	5 mL

200µL Omni-ATAC lysis buffer

50µL for each reaction, mix 200µL ATAC-RSB with 2µL 10% IGEPAL CA630 ,2µL 10 Tween 20 , 2µL 1% digitonin . Freshly prepare before use.

A	B	C
Reagents	200 µL	Final conc.
ATAC RSB	200 µL
10% IGEPAL CA630	2 µL	0.1%
10 % Tween 20	2 µL	0.1%
1% Digitonin	2 µL	0.01%

600µL Omni-ATAC wash buffer

150µL for each reaction, mix 600µL ATAC-RSB with 6µL 10% Tween 20. Freshly prepare before use.

A	B	C
Reagents	600 µL	Final conc.
ATAC RSB	600 µL
20% Tween 20	6 µL	0.1%

1mL 2 x TD buffer

mix 20µL 1 M TAPS pH 8.5, 10µL 1 M MgCl2, 200µL DMF , and bring the final volume to 1mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

A	B	C
Reagents	1 mL	Final conc.
1 M TAPS pH 8.5	20 µL	20 mM
1 M MgCl2	10 µL	10 mM
DMF	200 µL	20%
Nuclease-free H2O	770 µL

1mL 2 x STOP buffer

mix 80µL 0.5 M EDTA, 10µL 1 M Tris-HCl pH 8.0, 10µL 0.1M spermidine, and bring the final volume to 1mL with nuclease-free H2O. Freshly prepare before use.

A	B	C
Reagents	1 mL	Final conc.
0.5 M EDTA	80 µL	40 mM
1 M Tris-HCl pH 8.0	10 µL	10 mM
0.1 M spermidine	10 µL	1 mM
Total	1 mL

50µL Assemble META16 transposome METATAC_Primer_v.1.xlsx

1. Anneal META16 transposon

A	B
Oligos	Sequence
META16-1	GGCACCGAAAA
META16-2	CTCGGCGATAAA
META16-3	GGTGGAGCATAA
META16-4	CGAGCGCATTAA
META16-5	AGCCCGGTTATA
META16-6	TCGGCACCAATA
META16-7	GCCTGTGGATTA
META16-8	GCGACCCTTTTA
META16-9	GCATGCGGTAAT
META16-10	GCGTTGCCATAT
META16-11	GGCCGCATTTAT
META16-12	ACCGCCTCTATT
META16-13	CCGTGCCAAAAT
META16-14	TCTCCGGGAATT
META16-15	CCGCGCTTATTT
META16-16	CTGAGCTCGTTTT
19 bp ME	5'-/phos/-CTGTCTCTTATACACATCT-3'
META Tranposon	5'-[META sequence]-AGATGTGTATAAGAGACAG-3'

A	B	C
Reagents	Per 50 µL	Final conc.
10 x Annealing Buffer	5 µL	1x
50 µM META16 Transposon	5 µL	5 µM
50 µM 19 bp ME	5 µL	5 µM
H2O	35 µL

Mix thoroughly, then run the annealing program (95 °C, 1 min, gradual cooling, -0.1 °C /3s, 700 cycles to 25 °C, hold at 4 °C)

Recipe for 10x annealing buffer (500 mM NaCl, 100 mM Tris-HCl pH 8.0, 10 mM EDTA)

2. Assemble METAT16 transposome

Mix 25µL 5 µM Tn5 transposase and 25µL 5 µM annealed META16 transposon, incubate at 21-24°C for 30 min, protected from light.

Harvest fresh culture in a conical centrifuge tube (15 ml or 50 ml) at room temperature, centrifuge at 500 x g for 5 min at room temperature, then wash twice with 1x PBS pH 7.4, count cell number, stain with Trypan blue, and ensure viability >90%. then aliquot 50, 000 cells to a 200 µL PCR tube.

Pellet 50,000 viable cells at 500 x g at 4°C for 5 min in a swing bucket centrifuge, and remove supernatant carefully without disturbing the pellet.

Add 50 µL ice-cold Omni-ATAC lysis buffer (step 1.2), pipette up and down 10 times, then incubate on ice for 3 min.

Wash out lysis with 150 µL of ice-cold Omni-ATAC wash buffer (step 1.3) and invert the tube 3 times to mix.

Pellet nuclei at 500 x g for 10 min at 4°C in a swing bucket centrifuge.

Then wash one time with 50ul ice-cold Omni-ATAC wash buffer. Pellet nuclei at 500 x g at 4°C for 5min.

Transposition in Bulk

Prepare Transposition mix

A	B	C	D
Reagents	3 Rxn	Per Rxn	Final conc.
2 x TD buffer	37.5 µL	12.5 µL
META 16 Transposome	6 µL	2 µL	100 nM
1 x PBS	28.5 µL	9.5 µL
1% Digitonin	0.75 µL	0.25 µL	0.01%
10% Tween 20	0.75 µL	0.25 µL	0. 1%

Aspirate all supernatant, and avoid disrupting the visible pellet. Then resuspend the cell pellet in 25 µL of transposition mixture by pipetting up and down 10 times, then transfer to a 1.5 mL Lo-bind tube.

Incubate the reaction at 37°C for 30 minutes in a thermomixer with 1000 RPM mixing.

Add 25 µL 2x Stop buffer to stop transposition and incubate on ice for 10 min.

Add 50 µL 0.5% BSA (by dissolving 0.25 g BSA in 50 mL 1x PBS pH 7.4), then add 5 µL 7-AAD to stain nuclei.

FACS sort single 7-AAD positive nuclei to a 96-well PCR plate, containing 1 µL nuclei lysis buffer (10 mM Tris-HCl pH 8.0, 20 mM NaCl, 1 mM EDTA pH 8.0, 15 mM DTT, 0.1% SDS, 60 µg/mL QIAGEN protease).

Seal the plate with PCR sealing film (bio-rad, MSB1001), lysis was done by incubating at 65 °C for 15 min.

After lysis, add 1 µL 3% Triton X-100 to quench SDS. Spin down in a plate centrifuge, vortex to mix.

Amplification

Prepare preamplification mix

A	B	C	D
Reagents	120 Rxn	Per rxn	Final conc.
2 x High fidelity Q5 master mix	360	3	1x
50 µM META16 primer mix	23.04	0.192	100 nM each
100 mM MgCl2	6	0.05
Nuclease-free H2O	90.96	0.758
Cell lysate	NA	2	NA

A	B
Oligos	Sequence
META 16 Primer	5'-[META sequence]-AGATGTGTATAAG-3'

Aliquot 4 µL above preamplification mix to each well, Spin down in a plate centrifuge, vortex to mix.

Preamplification was incubated as

72°C, 5 min

98°C, 30 s

16 Cycles [98°C , 10 s; 62°C, 30 s; 72°C, 1 min]

72°C, 5 min

4°C hold.

Cell barcoding

A	B
Oligos	Sequence
META16-ADP1	5'-CTTTCCCTACACGACGCTCTTCCGATCT-[CB1]-[META sequence]-AGATGTGTATAAG-3'
META16-ADP2	5'-GAGTTCAGACGTGTGCTCTTCCGATCT-[CB2]-[META sequence]-AGATGTGTATAAG-3'
CB1-1	GATATG
CB1-2	ATACG
CB1-3	CCGTCTG
CB1-4	TGCG
CB1-5	GAACTCG
CB1-6	ATGTAG
CB1-7	CCCG
CB1-8	TATGT
CB1-9	GAGTAAG
CB1-10	ATCG
CB1-11	CCTAG
CB1-12	TGACCG
CB2-1	ACTCTA
CB2-2	AGAGCAT
CB2-3	GGTATG
CB2-4	TCGATGC
CB2-5	CTACTAG
CB2-6	TATGCA
CB2-7	CACACGA
CB2-8	GTCGAT

Add 0.45 µL of one of 96 barcode mixes to each well.

Incubate as

98°C, 30 s,

5 cycles [98°C, 10 s, 62°C, 30 s, 72°C, 1 min]

72°C, 5 min

Pool a whole plate for purification, typical 200 µL/plate for purification. DNA was extracted with ZYMO DCC5.

For fragment analysis, we use Agilent Fragment analyzer DNF474 kit, only samples with clear periodic nucleosome patterns are used for sequencing.

Library Preparation

Prepare Library prep mix

A	B	C	D
Reagents	40 Rxn	Per rxn	Final conc.
2x Q5 master mix	600	15	1x
NEB universal primer(10 µM)	20	2	0.67 µM
Neb i7 Index primer(10 µM)		2	0.67 µM
100 mM MgCl2	1	0.1
Template		10.9
Total		30

Library preparation was done by incubating as

98°C, 30 s

2 cycle [98°C, 10 s, 68°C, 30 s, 72°C, 1 min]

72°C, 5 min

Purify with ZYMO DCC5, then purify with 1.1 x SPRI beads to remove primer dimers.

For sequencing, we sequenced our sample on Illumina Hiseq 4000 or NovaSeq sequencer with 9 Gb/plate.

Raw read processing.

Raw Read Preprocessing. For both read 1 and read 2, the first 4 to 7 bases and the following 11 to 13 bases are paired cell barcodes and META sequence, respectively (step 1.6 attachment). We used a custom Python script to parse barcodes and split reads into individual fastq files for each cell, allowing up to one mismatch. Meanwhile, META sequences were annotated to read the name, allowing up to two mismatches. Then we used cutadapt to trim adapter sequences from both ends according to the 19-bp mosaic end (ME) sequence, with parameters -e 0.22 -a CTGTCTCTTATACACATCT and -e 0.22 -g AGATGTGTATAAGAGACAG for both read 1 and read 2. Processed reads were mapped to reference genome with bowtie2 -X 2000 –local –mm –no-discordant –no-mixed. hg38 (GRCh38, v26) reference genome was used for human cells, and mm10 (GRCm38, vM19) reference genome was used for mouse cells. Reads with mapping quality of less than 30 were filtered out from the further analysis. PCR duplicates were identified and removed with a custom script, according to their positions on the genome and META tags. Paired-end reads were converted to fragments with Tn5 insertion centering correction (R1 start +4 and R2 end 5). Finally, for each cell, contaminated fragments from other cells were removed based on the aligned coordinates, META sequences, and read frequency.