Sep 30, 2022

Public workspaceMETATAC V.1 V.2

  • Honggui Wu1,
  • Xiang Li2,1,3,
  • Fanchong Jian2,1,4,
  • Ayijiang Yisimayi2,1,5,
  • X. Sunney Xie2,1,6
  • 1Biomedical Pioneering Innovation Center, Peking University, Beijing 100871, China;
  • 2Beijing Innovation Center for Genomics, Peking University, Beijing 100871, China;
  • 3Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China;
  • 4College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PRC;
  • 5School of Life Sciences, Peking University, Beijing, 100871, China;
  • 6School of Life Sciences, Peking University, Beijing 100871, China Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
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Protocol CitationHonggui Wu, Xiang Li, Fanchong Jian, Ayijiang Yisimayi, X. Sunney Xie 2022. METATAC V.1. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg36x51g25/v2Version created by Honggui Wu
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 30, 2022
Last Modified: September 30, 2022
Protocol Integer ID: 70695
Keywords: scATAC-seq, chromatin accessibility, single-cell epigenomics,
Funders Acknowledgement:
the Beijing Municipal Commission of Science and Technology
Grant ID: Z201100005320015
Abstract
Abstract
Here we describe a protocol for multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), a high-sensitivity single-cell ATAC-seq technique with the help of META chemistry and extensive biochemical modifications. We improved the protocol from three aspects, first, we used Omni-ATAC protocol to permeabilize cells, which greatly reduced mitochondrial reads to less than 0.5%. Second, we used sodium dodecyl sulfate (SDS) to release Tn5 from bound DNA, which enabled maximum DNA recovery. Third, we use META transposome instead Nextera transposome used in other protocols, which avoids half loss due to self-looping during amplification, thus further increasing library size. Specifically, META sequences also serve as a barcode to fragment decontamination, which provides high-sensitivity and precise single-cell chromatin accessibility analysis.
Protocol materials
ReagentDigitonin, 40ulPromegaCatalog #G9441
Step 1.2
ReagentNN-Dimethylformamide anhydrousMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4551-250ML
Step 1.4
ReagentIGEPAL CA-630Merck MilliporeSigma (Sigma-Aldrich)Catalog #I8896-50ML
Step 1.2
ReagentTween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Step 1.2
REAGENT SETUP
REAGENT SETUP
1h 30m
1h 30m
Prepare METATAC Reagents
1h 30m
Amount5 mL ATAC resuspension buffer (ATAC-RSB)
Mix Amount50 µL 1 M Tris-HCl pH 7.5, Amount10 µL 5 M NaCl, Amount15 µL 1 M MgCl2, and bring the final volume to Amount5 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

ABC
Reagents5 mLFinal conc.
1 M Tris-HCl pH 7.550 µL10 mM
5 M NaCl10 µL10 mM
1 M MgCl215 µL3 mM
Nuclease-free H2O4925 µL
Total5 mL




Amount200 µL Omni-ATAC lysis buffer
Amount50 µL for each reaction, mix Amount200 µL ATAC-RSB with Amount2 µL 10% IGEPAL CA630ReagentIGEPAL CA-630Sigma AldrichCatalog #I8896-50ML ,Amount2 µL 10 Tween 20ReagentTween 20Bio-rad LaboratoriesCatalog #170-6606-MSDS , Amount2 µL 1% digitoninReagentDigitonin, 40ulPromegaCatalog #G9441 . Freshly prepare before use.


ABC
Reagents200 µLFinal conc.
ATAC RSB200 µL
10% IGEPAL CA6302 µL0.1%
10 % Tween 202 µL0.1%
1% Digitonin2 µL0.01%


Amount600 µL Omni-ATAC wash buffer
Amount150 µL for each reaction, mix Amount600 µL ATAC-RSB with Amount6 µL 10% Tween 20. Freshly prepare before use.

ABC
Reagents600 µLFinal conc.
ATAC RSB600 µL
20% Tween 206 µL0.1%


Amount1 mL 2 x TD buffer
mix Amount20 µL 1 M TAPS pH 8.5, Amount10 µL 1 M MgCl2, Amount200 µL DMFReagentNN-Dimethylformamide anhydrousSigma AldrichCatalog #D4551-250ML , and bring the final volume to Amount1 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

ABC
Reagents1 mLFinal conc.
1 M TAPS pH 8.520 µL20 mM
1 M MgCl210 µL10 mM
DMF200 µL20%
Nuclease-free H2O770 µL


Amount1 mL 2 x STOP buffer
mix Amount80 µL 0.5 M EDTA, Amount10 µL 1 M Tris-HCl pH 8.0, Amount10 µL 0.1M spermidine, and bring the final volume to Amount1 mL with nuclease-free H2O. Freshly prepare before use.

ABC
Reagents1 mLFinal conc.
0.5 M EDTA80 µL40 mM
1 M Tris-HCl pH 8.010 µL10 mM
0.1 M spermidine10 µL1 mM
Total1 mL


Amount50 µL Assemble META16 transposomeDownload METATAC_Primer_v.1.xlsxMETATAC_Primer_v.1.xlsx
1) Anneal META16 transposon

AB
OligosSequence
META16-1GGCACCGAAAA
META16-2CTCGGCGATAAA
META16-3GGTGGAGCATAA
META16-4CGAGCGCATTAA
META16-5AGCCCGGTTATA
META16-6TCGGCACCAATA
META16-7GCCTGTGGATTA
META16-8GCGACCCTTTTA
META16-9GCATGCGGTAAT
META16-10GCGTTGCCATAT
META16-11GGCCGCATTTAT
META16-12ACCGCCTCTATT
META16-13CCGTGCCAAAAT
META16-14TCTCCGGGAATT
META16-15CCGCGCTTATTT
META16-16CTGAGCTCGTTTT
19 bp ME5'-/phos/-CTGTCTCTTATACACATCT-3'
META Tranposon5'-[META sequence]-AGATGTGTATAAGAGACAG-3'


ABC
Reagents Per 50 µLFinal conc.
10 x Annealing Buffer 5 µL 1x
50 µM META16 Transposon5 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 Amount25 µL 5 µM Tn5 transposase and Amount25 µL 5 µM annealed META16 transposon, incubate at Temperature21-24 °C for 30 min, protected from light.

Note
Assembled transposome can be stored at -80 °C for up to six months. Tn5 transposase was expressed in-house from the plasmid pTXB1-Tn5 or purchased from Vazyme (s111-01).


Assembled META16 transposome at a final concentration of 1.25 µM dimer.

Bulk Transposition
Bulk Transposition
1h 30m
1h 30m
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.


Note
In order to have enough nuclei for FACS, we recommend preparing 2-3 replicates, which is enough for 50-60 96-well plates.



Note
Our protocol also works well for cryopreserved samples and nuclei. For the cryopreserved sample, quickly thaw one tube of the cell at 37 °C water bath, then wash once with ice-cold PBS, count cell number, aliquot 50,000 each.


20m
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.
6m
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.


Note
The incubation time depends on cell type, 3 min works well for most samples, but we found for the digestive system sample, incubation needs to extend to 5-10 min to get the periodic nucleosomal pattern.


4m
Wash out lysis with 150 µL of ice-cold Omni-ATAC wash buffer (step 1.3) and invert the tube 3 times to mix.
2m
Pellet nuclei at 500 x g for 10 min at 4°C in a swing bucket centrifuge.
11m
Then wash one time with 50ul ice-cold Omni-ATAC wash buffer. Pellet nuclei at 500 x g at 4°C for 5min.
8m
Transposition in Bulk
50m
Prepare Transposition mix
ABCD
Reagents 3 Rxn Per Rxn Final conc.
2 x TD buffer 37.5 µL12.5 µL
META 16 Transposome 6 µL2 µL100 nM
1 x PBS28.5 µL9.5 µL
1% Digitonin 0.75 µL0.25 µL0.01%
10% Tween 20 0.75 µL0.25 µL0. 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.
FACS single nuclei
FACS single nuclei
1h
1h
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).

Note
If not proceed immediately, seal the plate with Aluminum Sealing Film (Axygen, PCR-AS-600), and store at -80°C for several months without lysis.


Note
All liquid transfer steps can be done with a multichannel pipette or with an automated liquid handler system (e.g., Beckman Biomek FXP liquid handler, Echo 525 acoustic liquid handler system). Here, we use Echo 525 to handle all the liquid transfer steps, which process 20 plates each time.

For cell lysis buffer, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 45 s/plate.

Amplification
Amplification
3h 30m
3h 30m
Seal the plate with PCR sealing film (bio-rad, MSB1001), lysis was done by incubating at 65 °C for 15 min.
30m
After lysis, add 1 µL 3% Triton X-100 to quench SDS. Spin down in a plate centrifuge, vortex to mix.


Note
For 3% Triton X-100, we use Echo 525 to aliquot with 384PP_AQ_SPHigh calibration. 45 s/plate.

20m
Amplification
2h
Prepare preamplification mix
ABCD
Reagents 120 RxnPer rxnFinal conc.
2 x High fidelity Q5 master mix3603 1x
50 µM META16 primer mix23.040.192100 nM each
100 mM MgCl2 60.05
Nuclease-free H2O90.960.758
Cell lysate NA 2 NA

AB
OligosSequence
META 16 Primer5'-[META sequence]-AGATGTGTATAAG-3'


Note
META16 primer sequences see above .

20m
Aliquot 4 µL above preamplification mix to each well, Spin down in a plate centrifuge, vortex to mix.


Note
For the preamplification mix, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 2 min/plate.

45m
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.
50m
Cell barcoding

Note
Cell barcoding was realized using a 12 x 8 barcode combination. Premix 12 META16-ADP1 barcodes and 8 META16-ADP2 barcodes into 96 barcode combinations. Detailed sequences see below table.



AB
OligosSequence
META16-ADP15'-CTTTCCCTACACGACGCTCTTCCGATCT-[CB1]-[META sequence]-AGATGTGTATAAG-3'
META16-ADP25'-GAGTTCAGACGTGTGCTCTTCCGATCT-[CB2]-[META sequence]-AGATGTGTATAAG-3'
CB1-1GATATG
CB1-2ATACG
CB1-3CCGTCTG
CB1-4TGCG
CB1-5GAACTCG
CB1-6ATGTAG
CB1-7CCCG
CB1-8TATGT
CB1-9GAGTAAG
CB1-10ATCG
CB1-11CCTAG
CB1-12TGACCG
CB2-1ACTCTA
CB2-2AGAGCAT
CB2-3GGTATG
CB2-4TCGATGC
CB2-5CTACTAG
CB2-6TATGCA
CB2-7CACACGA
CB2-8GTCGAT


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


Note
For the cell barcode, we use Echo 525 to aliquot with 384PP_AQ_BP calibration, 30 s/plate.

40m
Pool a whole plate for purification, typical 200 µL/plate for purification. DNA was extracted with ZYMO DCC5.
It's a typical amplicon of GM12878 cells, but the fragment size distribution varies according to different cell types.

1h 30m
For fragment analysis, we use Agilent Fragment analyzer DNF474 kit, only samples with clear periodic nucleosome patterns are used for sequencing.

Wxamplified library showed good periodic nucleosome pattern.

Examplified library showed poor periodic nucleosome pattern.

Library preparation
Library preparation
Library Preparation

1h 30m
Prepare Library prep mix

ABCD
Reagents40 RxnPer rxnFinal conc.
2x Q5 master mix600151x
NEB universal primer(10 µM)2020.67 µM
Neb i7 Index primer(10 µM) 20.67 µM
100 mM MgCl2 10.1
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

Note
2 cycles of amplification are critical to avoid residual cell barcode primers in purified amplicons causing cell-to-cell contamination.

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

METATAC Library schematic diagram

Sequencing
Sequencing
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.