TITLE: METATAC V.1
AUTHORS: Honggui Wu, Xiang  Li, Fanchong Jian, Ayijiang  Yisimayi, X.  Sunney Xie

[DESCRIPTION]
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.

[STEPS]
SECTION: Bulk Transposition
2.  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.


 



 


SECTION: Bulk Transposition
3. 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.
SECTION: Bulk Transposition
4. 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. 


 


SECTION: Bulk Transposition
5. Wash out lysis with 150 µL of ice-cold Omni-ATAC wash buffer (step 1.3) and invert the tube 3 times to mix. 
SECTION: Bulk Transposition
6. Pellet nuclei at 500 x g for 10 min at 4°C in a swing bucket centrifuge.
SECTION: Bulk Transposition
7. Then wash one time with 50ul ice-cold Omni-ATAC wash buffer. Pellet nuclei at 500 x g at 4°C for 5min.
SECTION: Bulk Transposition
8. Transposition in Bulk
SECTION: Bulk Transposition
8.1. Prepare Transposition mix
 
     Components      2 Rxn      Per Rxn      Final con        2 x TD buffer      25      12.5               META 16 Transposome             2               PBS      18.75      9.375               1% Digitonin      0.5      0.25      0.01%        10% Tween      0.5      0.25      1%   

SECTION: Bulk Transposition
8.2. 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. 
SECTION: Bulk Transposition
8.3. Incubate the reaction at 37°C for 30 minutes in a thermomixer with 1000 RPM mixing.
SECTION: Bulk Transposition
8.4. Add 25 µL 2x Stop buffer to stop transposition and incubate on ice for 10 min.
SECTION: FACS single nuclei
9. 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.
SECTION: FACS single nuclei
10. 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).

 


 

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


 

SECTION: Amplification
13. Amplification
 
SECTION: Amplification
13.1. Prepare preamplification mix
 
     Component      900   Rxn x 3      Per   rxn      Final   con        2x   Q5 master mix      2700      3      1x        META   16 primer mix      172.8      0.192      100nM   each        100mM   MgCl2      45      0.05               H2O      682.2      0.758               Cell   lysate      NA      2      NA        Total      3600   x 3      6      NA   

 

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


 

SECTION: Amplification
13.3. 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.
SECTION: Amplification
14. Cell barcoding

 

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


 

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

SECTION: Library preparation
17. Library Preparation

SECTION: Library preparation
17.2. 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

 

SECTION: Library preparation
17.3. Purify with ZYMO DCC5, then purify with 1.1 x SPRI beads to remove primer dimers.
 

SECTION: REAGENT SETUP
1. Prepare METATAC Reagents
SECTION: REAGENT SETUP
1.1. 5 mL ATAC resuspension buffer (ATAC-RSB)
Mix 50 µL  1 M Tris-HCl pH 7.5, 10 µL 5 M NaCl, 15 µL 1 M MgCl2, and bring the final volume to 5 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

 




SECTION: REAGENT SETUP
1.2. 200 µL Omni-ATAC lysis buffer
50 µL  for each reaction, mix 200 µL  ATAC-RSB with 2 µL 10% IGEPAL CA630  ,2 µL 10 Tween 20  , 2 µL 1% digitonin  . Freshly prepare before use.


 


SECTION: REAGENT SETUP
1.3. 600 µL Omni-ATAC wash  buffer
150 µL for each reaction, mix 600 µL  ATAC-RSB with 6 µL 10% Tween 20. Freshly prepare before use.

 


SECTION: REAGENT SETUP
1.4. 1 mL 2 x TD buffer
mix 20 µL 1 M TAPS pH 8.5, 10 µL 1 M MgCl2, 200 µL DMF  , and bring the final volume to 1 mL with nuclease-free H2O. Store the buffer at -20 °C for up to several months.

 


SECTION: REAGENT SETUP
1.5. 1 mL 2 x STOP buffer
mix 80 µL 0.5 M EDTA, 10 µL 1 M Tris-HCl pH 8.0, 10 µL 0.1M spermidine, and bring the final volume to 1 mL with nuclease-free H2O. Freshly prepare before use.

 


SECTION: REAGENT SETUP
1.6. 50 µL Assemble META16 transposome  
1) Anneal META16 transposon
     Components      Per 50ul      Final concentration        10 x Annealing Buffer      5      1x        100uM META16 Adapter      2.5      5uM        50uM 19bp ME      5      5uM        H2O      37.5          
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 25 µL 5 µM Tn5 transposase and 25 µL 5 µM annealed META16 transposon, incubate at 21-24 °C  for 30 min, protected from light. 

 

SECTION: Library preparation
18.   

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

    

  

SECTION: Sequencing
19. For sequencing, we sequenced our sample on Illumina Hiseq 4000 or NovaSeq sequencer with 9 Gb/plate. 
SECTION: Library preparation
17.1. Prepare Library prep mix

 

SECTION: Sequencing
20. 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.