alias: GSM2889305
title: GSM2889305: shSetdb1 rep#2 (ATAC-seq); Mus musculus; ATAC-seq
library_construction_protocol: ATAC-seq was performed as described (Buenrostro et al., 2015). In brief, a total of ~50,000 cells were washed once with 50 μl of cold PBS and resuspended in 50μl lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase and 22.5 μl nuclease-free H2O) of Nextera DNA library Preparation Kit (96 samples) (FC-121-1031, Illumina). Samples were then PCR amplified and incubated at 37°C for 30min. DNA was isolated using a MinElute Kit (Qiagen). ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described (Buenrostro et al., 2015), and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with a Qiaquick PCR (Qiagen) column and were quantified using a KAPA Library Quantification kit (KK4824, Kapa Biosystems) according to the manufacturer's instructions. Library integrity was checked by gel electrophoresis. Finally, the ATAC library was sequenced on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina) according to the manufacturer's instructions. According to the extract protocol
identifiers.namespace: GEO
identifiers.id: GSM2889305