GSM3676132: WT_cells_d5_rep1 (ATAC-seq); Mus musculus; ATAC-seq
The protocol was modified from Buenrostro et al. EBs were trypsinized to single-cell suspension, washed with PBS, lysed and mixed with 50 µl of transposition reaction mix containing Tagment DNA Enzyme (Nextera DNA Library Preparation Kit, Illumina) and 0.2% Digitonin. The transposition reaction was carried out at 37°C for 30 min, purified using the Qiagen MinElute Kit and eluted in EB-buffer (Qiagen). DNA was amplified by PCR and purified with the Qiagen MinElute Kit (Qiagen, 28004). Libraries were prepared using Nextera DNA Library Preparation Kit (New England BioLabs). The libraries were size-selected by polyacrylamide gel electrophoresis (PAGE) by loading the samples onto a 6% polyacrylamide gel and run in TBE buffer (89 mM Tris-HCl, 89 mM Boric acid, 2 mM Na2EDTA in H2O, pH 8.0). DNA was visualized with SYBRTMTM Gold Nucleic Acid Gel Stain (Invitrogen, 1:10,000 dilution in TBE-buffer). Gel containing fragments between 100-1,000 bp was cut and resuspended in Diffusion buffer (0.5 M CH3COONH4, 10 mM Mg(CH3COO)2, 1 mM Na2EDTA, 0.1% SDS in H2O) in a 1:1 ratio of buffer to gel (mg). After incubation at 37°C for 22 h samples were filtered using a 30 µm CellTrics strainer and purified using the QIAquick Gel Extraction Kit (Qiagen).
GEO Accession
GSM3676132