Ten(10)X-compatible Combinatorial Indexing ATAC sequencing (txci-ATAC-seq)

Resuspend Tn5ME-A, Tn5ME-B, and Tn5MErev in the annealing buffer to a final concentration of 100 μM.

Prepare annealed linker A: Mix one volume of Tn5ME-A with one volume of Tn5MErev in a PCR tube.

e.g. 100 μl Tn5ME-A + 100 μl Tn5Merev.

Prepare annealed linker B: Mix one volume of each barcoded Tn5ME-B with one volume of Tn5MErev on a 96-well plate.

e.g. 10 μl Tn5ME-B (Index A1) + 10 μl Tn5MErev.

Table1_Barcoded_Tn5MEB.xlsx

Mix briefly by pipetting and run the following PCR program in a thermocycler for annealing oligos.

A	B
95 ℃	5 min
Slowly Cool down to 65 ℃	-0.1 ℃/sec
65 ℃	5 min
Slowly Cool down to 4 ℃	-0.1 ℃/sec

The annealed oligos can be kept at -20°C for long-term storage.

Add 1 μl of each annealed linker (A and B) to 20 μl of the Tn5 stock (0.3 mg/ml) on a 96-well plate with a unique annealed linker B in each well.

Mix briefly by pipetting, and then incubate at 23°C for 30 minutes in a thermomixer at 350 rpm.

Store at -20°C.

Remove approximately 10x10⁶cells from culture.

Pellet the cells at 500 RCF at 4°C for 5 min in a swinging-bucket centrifuge.

Aspirate supernatant.

Resuspend pellet in 200 µl RSB Lysis Buffer.

Incubate on ice for 3 minutes.

Add 1 ml RSB Washing Buffer.

Take 10 µl nuclei and dilute it with 40 µl of Omni TD buffer, then count the nuclei on a hemocytometer by adding 50 µl Trypan blue solution to the diluted nuclei (Note: we found that adding the RSB-resuspended nuclei straight to Trypan blue solution will cause inflation of nuclei, and diluting nuclei in Omni TD buffer before exposure to Trypan blue improves the nuclei integrity).

Pellet the remaining nuclei in RSB Washing Buffer at 500 RCF for 10 min at 4°C in a fixed-angle centrifuge.

Resuspend nuclei pellet in FBW at ~3 million nuclei/ml.

Snap-freeze nuclei in liquid nitrogen, and then transfer the cryovials to a liquid nitrogen dewar (or -80°C) for long-term storage.

The following protocol can be used to isolate nuclei from lung tissues.

Take out flash-frozen nuclei (~3 million in 1 ml) from liquid nitrogen for each sample and thaw in a water bath at 37°C for about 1 min.

Add 3 ml RSB washing buffer to an empty 15 ml tube for each sample.

Transfer 1 ml nuclei stored in the freezing buffer to the 15 ml tube containing 3 ml RSB washing buffer.

Pellet the nuclei at 500 RCF for 10 min at 4°C.

Resuspend nuclei with 1 ml RSB washing buffer and then transfer to a 1.5 ml LoBind tube through Flowmi (40 micron).

Pellet the nuclei at 500 RCF for 5 min at 4°C in a fixed-angle centrifuge.

Resuspend nuclei with 100 μl of 1X PBSB for each sample.

Count nuclei with DAPI:

Add 1 μl of 300 μM DAPI to 100 ul nuclei;

Incubate on ice for 5 mins;

Add 10 μl stained nuclei to the countess slide to count nuclei.

Prepare TD mix:

A	B	C	D
2X Nextera TD Buffer*	1X	12.5 µl	1500
1% Digitonin	0.01%	0.25 µl	30
10% Tween-20	0.1%	0.25 µl	30

*Omni TD buffer can be used to replace the Illumina Nextera TD buffer.

.

Thaw a 96-well plate preloaded with 5 μl of barcoded Tn5 on ice. Mix by brief shaking at 1400 rpm for 30 seconds, spin for a minute at 2000 RCF at 4°C, and carefully unseal the aluminum foil seal.

Dilute nuclei to 2857 nuclei/μl in PBSB and then mix 7 μl diluted nuclei with 13 μl TD mix for each well.

Add 20 μl nuclei/TD mix mixture to each well of the 96-well plate containing 5 μl of barcoded Tn5 per well (total 25 μl).

Seal the plate using Bio-Rad Microseal B film.

Mix by shaking at 1000 rpm for one minute.

If liquid splashes to the seal, briefly spin at 500 RCF for 10 sec.

Incubate at 37°C for 60 min in a thermocycler block with a heated lid (47°C).

Thaw TMG washing buffer on ice.

Remove the plate from the thermocycler.

Briefly centrifuge at 500 RCF for 10 sec at 4°C.

Incubate the plate on ice for 5 min.

Pool nuclei in a LoBind 12-tube strip and then transfer them to a 15 ml conical tube preloaded with 400 ul of TMG.

Add 50 μl/well of TMG to the first row of the plate and pipette them throughout the whole plate to wash out the residual nuclei remaining in the plate.

After washing the last row of the plate, the TMG was transferred to the same conical tube that was used to collect the barcoded nuclei.

Centrifuge nuclei at 500 RCF for 10 min at 4°C.

Remove most of the supernatant.

Resuspend nuclei with 500 μl of TMG, then transfer to a 1.5 ml LoBind Tube through Flowmi.

Centrifuge at 500 RCF for 5 min at 4°C.

Remove most of the supernatant and resuspend the nuclear pellet with 30 μl of LBS.

Count nuclei with a hemocytometer.

Take the volume of solution containing the desired number of nuclei and dilute it with the LBS to make a total of 15 μl.

Use the 15 μl dilution as input into the 10X Chromium droplet generator – follow Step 2, page 24 of the Chromium Single Cell ATAC kit instructions (10x Document CG000209 Rev D) to complete the assay.

For Step 2.5. GEM Incubation:

a. Incubate in a thermal cycler with the following protocol (Lid temperature at 105℃).

A	B
72 ℃	00:05:00
98 ℃	00:00:30
98 ℃	00:00:10
59 ℃	00:00:30
72 ℃	00:01:00; Go to step 3, repeat 7X (Total 8 cycles)
15 ℃	Hold

b. Store at 15°C for up to 18 h or at −20°C for up to 1 week, or proceed to the next step.

For Step 4.1 Sample Index PCR

c. Add 2.5 μl of customized i7 TruSeq primer (25 μM) containing an 8 bp custom barcode to each 10X library. Record assignment. Pipette mix and centrifuge briefly.

Table2_TrueSeq_i7_Primer.xlsx

d. Incubate in a thermal cycler with the following protocol (Lid temperature at 105℃).

A	B
98 ℃	00:00:45
98 ℃	00:00:20
67 ℃	00:00:30
72 ℃	00:00:20; Go to step 2, repeat 4X (Total 5 cycles)
72 ℃	00:01:00
4 ℃	Hold

A	B
Read 1N	51 bp
i7 index (I1)	8 bp
i5 index (I2)	16 bp
Read 2N	78 bp