title: GSM5290921: GM12878-Fixed-20k-Omni_TD-37C-Homemade_Tn5-Rep4; Homo sapiens; ATAC-seq
library_construction_protocol: 1) Nuclei isolation from native GM12878:  ~2 million native cells were collected and washed using 4 ml 1X PBS (pH 7.4, Gibco, cat. no. 10-010-023) supplemented with 0.04% BSA (PBSB), and then resuspended in 200 μl of ATAC-seq lysis buffer. Lysis buffer was made by supplementing ATAC resuspension buffer (RSB) with detergents. RSB buffer is 10 mM Tris-HCl (pH 7.5, Invitrogen, cat. no. 15567027), 10 mM NaCl (Invitrogen, cat. no. AM9759) and 3 mM MgCl2 (Invitrogen, cat. no. AM9530G) in nuclease free water. RSB was made in bulk and stored at 4°C long-term. On the day of the experiment, the ATAC lysis buffer was made by adding 0.1% IGEPAL (Sigma, cat. no. I3021), 0.01% digitonin (Invitrogen, cat. no. BN2006), and 0.1% Tween-20 (Bio-Rad, cat. no. 1610781) to RSB. Detergent percentages reported are final concentrations. After resuspending cell pellets in the lysis buffer, they were incubated on ice for 3 min, and then the lysis was stopped by adding 1 ml RSB containing 0.1% Tween-20. Nuclei were then centrifuged at 500 r.c.f for 10 min at 4 °C and resuspended in 200 ul of PBSB. The nuclei were counted and diluted to 3,100 nuclei/μl with PBSB. Then, 6.6 μl of diluted nuclei (20,460 total nuclei) were transferred to 12.4 μl of transposition reaction (10 μl of 2X Tagmentation buffer, 0.2 μl of 1% digitonin, 0.2 μl of 10% Tween-20, and 2 μl of H2O). We then added 1 μl of transposase to the transposition mix containing nuclei. Tagmentation was carried out on a thermocycler at either 37°C or 55°C for 30 min. Tagmented DNA was cleaned up using Zymo DNA Clean and Concentrator-5 columns with 5X binding buffer (Zymo, cat. no. D4004). 2) Nuclei isolation from fixed GM12878: the frozen, fixed cells (~ 2 million) were incubated in a 37°C water bath until thawed (~ 1 min). After thawing, the cells stored in 1 ml freezing buffer were diluted with 3 ml of PBSB. The fixed cells were collected by centrifuging at 500 r.c.f for 10 min at 4°C, resuspended again in 1 ml PBSB, and then centrifuged at 500 r.c.f for 5 min at 4°C to wash the cells. Following washing, the nuclei were isolated and tagmented as described in the ATAC-seq protocol for native samples described above. After tagmentation, crosslinks were then reversed for the fixed, tagmented nuclei by adding 60 μl of reverse-crosslinking buffer containing 0.067% SDS and 1.33 mg/ml Proteinase K (Qiagen, cat. no. 19133) in Qiagen Buffer EB (final concentration of 0.05% SDS and 1 mg/ml Proteinase K) directly to the tagmentation reactions, and incubating the samples at 65°C for 15 hours in a thermomixer with shaking at 1,000 r.p.m. Following crosslink reversal, tagmented DNA was purified using Zymo DNA Clean and Concentrator-5 columns with 5X binding buffer. 3) The isolation of mouse lung nuclei was performed following the single-nucleus isolation protocol described by Joshi Nikita and Misharin Alexander. In brief, we cut a ~0.1-0.2 g piece from the lung sample removed from -80°C and kept it on dry ice until use. The tissue block was thawed almost completely on ice for 1 min, and then injected with 1 ml of cell lysis buffer, which was made of 1x cOmplete protease inhibitor cocktail (1 tablet per 10 ml solution, Sigma-Aldrich, Cat. 11836153001) in Nuclei EZ prep buffer (Sigma-Aldrich, Cat. NUC101), into the center of the tissue with a 30G needle and syringe. Following lysis buffer injection, the tissue was chopped into small pieces with scissors and then transferred along with the lysing buffer into a gentleMACS C tube (Miltenyi Biotec, Cat. 130-096-334). An additional 1 ml of lysing buffer was added into the C tube to make a final volume of 2 ml. The minced tissue was then homogenized using a gentleMACS tissue dissociator by running the 'm_lung_01' program followed by the first 20 sec of the 'm_lung_02' program. After homogenization, tissue lysate was briefly centrifuged to reduce foam and then passed through a 40 μm cell strainer in a 50ml tube. After passing the sample through, the strainer was rinsed with 4 ml of washing buffer (PBS with 1% BSA). The nuclei were counted with Trypan blue in the presence of 2X Omni buffer (20 mM Tris HCl (pH 7.5), 10 mM MgCl2 and 20% Dimethyl Formamide), and then collected by centrifugation at 500 r.c.f for 5 min at 4°C. Then, we removed the supernatant and resuspended the nuclei to a concentration of 4-5 million nuclei/ml in a nuclei freezing buffer containing 50 mM Tris-HCI (pH 8.0, Invitrogen, cat. no. 15568025), 5 mM Magnesium Acetate (Sigma, cat. no. 63052), 25% glycerol (VWR, cat. no. RC3290-32), 0.1 mM EDTA (Fisher, cat .no. AM9260G), 5 mM DTT (Fisher, cat. no. P2325), and 2% protease inhibitor (Sigma, cat. no. P8340). 1 ml aliquots of the nuclei were flash frozen in liquid nitrogen and then transferred to a liquid nitrogen dewar for long-term storage. 5 μl of purified DNA out of 10 μl purified product was amplified in a 25 μl PCR reaction containing NEBNext PCR master mix (1X final), 0.5X SYBR Green and 1.25 μM of Ad1 primer from Buenrostro JD. et al., 2013 and 1.25 μM of an N7 primer containing custom barcodes. Samples were amplified on a Bio-Rad CFX Connect Real-time cycler using the following program: 72°C for 5 min; 98°C for 30s; cycling at 98°C for 10 s, 63°C for 30 s, 72°C for 1 min; samples were monitored and stopped when it appeared that their exponential amplification was leveling off. At this point the samples were allowed to incubate at 72°C for an additional minute to fully extend the library. All native samples were stopped after 9 cycles and fixed samples were stopped after 10 cycles. PCR products were cleaned up with Ampure XP beads. A double size selection was performed to remove DNA fragments larger than ~1500 bp (with 0.4X AMPure XP beads) and smaller than ~100 bp (with 1.5X AMPure XP beads). To do this, we added 25 μl of Qiagen Buffer EB to each PCR reaction to bring the volume to 50 μl, and then added 20 μl of beads (homogenizing the mixture well by pipetting), followed by a 5 min incubation at RT. The samples were then put on a magnet stand to bind the beads. 68 μl of the supernatant was transferred to a new 1.5 ml tube and an additional 53 μl of beads were added to the supernatant and resuspended thoroughly (we estimated that 19.4 μl of the 68 μl transferred was bead buffer and 48.6 μl was sample, so to get to 1.5X beads for the second selection we needed to add an additional 72.9 - 19.4 μl of beads, which we rounded off to 53 μl). After another 5 min at RT, the samples were placed on the magnet to clear the beads and the supernatant was discarded. Beads were washed twice with 200 μl of 80% EtOH (made fresh for each experiment). After the second wash, the beads were briefly spun, and any residual ethanol was removed. The beads were then put back on the magnet stand and air dried for 1 min. Then, beads were removed from the magnet and resuspended in 22 μl of Buffer EB, incubated for 2 min at RT and then placed on the magnet stand again. 20 μl of supernatant was transferred to a new tube. The 96 ATAC-seq libraries of the GM12878 cell line and 72 libraries of mouse lungs were pooled separately and sequenced with 2x76 bp reads in two independent runs on an NextSeq 550 Platform using the High Output Kits.