GSM5501693: PD4_ATAC_WT_Rep2; Mus musculus; ATAC-seq
Freshly FACS-sorted CD140a+CD26+ cells from mouse dermis were subjected to ATAC-seq and ChIP-seq library preparation. For ATAC-seq, cells were incubated with cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), then were immediately subjected to a transposition reaction for 30 min at 37°C with TN5 transposase (Illumina). Transposed DNA was purified using Qiagen MiniElute purification kit and PCR amplified with 10-15 cycles. Library construction was performed using Nextera primers (Illumina). The libraries were sequenced using Illumina HiSeq 2500 platform using a 50-bp paired-end setting. ATAC-seq experiments were independently repeated for two times.  For ChIP-seq, library preparation was performed using ChIPmentation method (Nat Methods 12, 963–965 (2015).). Briefly, cells were fixed with 1% paraformaldehyde in PBS for 10 min at 25C, then were collected at 500 g for 10 min at 4°C and washed twice with 1 μM phenylmethyl sulfonyl fluoride (PMSF) in PBS. The pellet was sonicated a Branson 102C sonicator on ice until the size of fragments was in the range of 200–700 bp. The lysate was centrifuged and the supernatant containing the sonicated chromatin was transferred to a new tube. The sonicated lysate was brought to in 200 μl RIPA buffer per immunoprecipitation. For each immunoprecipitation, 10 μl Dynabeads Protein A were washed twice and resuspended with 0.1% BSA in PBS. The antibody was added and bound to the beads by rotating for 2 h at 4 °C. Anti-H3K27ac antibody (1 μg per immunoprecipitation, Diagenode pAB-196-050) and a nonspecific IgG rabbit antibody (2.5 μg per immunoprecipitation, Millipore 12-370) were used. Blocked antibody-conjugated beads were then placed on a magnet, supernatant was removed, and the sonicated lysate was added to the beads followed by incubation by rotating for 3 h at 4°C. Beads were washed subsequently with RIPA, RIPA-HS and RIPA-LiCl buffers. Beads were washed with cold Tris-HCl pH 8.0 and then resuspended in 25 μl tagmentation reaction buffer (10 mM Tris pH 8.0, 5 mM MgCl2, 10% v/v dimethylformamide) containing 1 μl TN5 transposase enzyme (Illumina) and incubated for 5 min at 37 °C in a thermocycler. Beads were washed with RIPA and cold Tris-HCl pH 8.0 and then incubated with 70 μl elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.4% SDS) containing 2 μl of Proteinase K (NEB) for 1 h at 55 °C and for 6 h at 65 °C, and supernatant was transferred to a new tube. Transposed DNA was purified with Qiagen MiniElute purification kit, and PCR amplified using Kapa HiFi HotStart ReadyMix (Kapa Biosystems). Enriched libraries were purified using SPRI AMPure XP beads at a beads-to-sample ratio of 1:1, followed by a size selection using AMPure XP beads to recover libraries with a fragment length of 200–400 bp. Library construction was performed using TruSeq ChIP Library Preparation Kit (Illumina). The libraries were sequenced using Illumina HiSeq 2500 platform using a 100-bp paired-end setting.