title: GSM2991101: ATAC_control; Mus musculus; ATAC-seq
library_construction_protocol: Cells from ATAC-seq libraries created from approximately fifty 8-cell embryos were prepared as described previously (Wu et al., 2016). Briefly, samples were lysed in 5 μL of lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and 0.15% NP-40) for 10 min on ice. Immediately after lysis, samples were then incubated with the Tn5 transposase and tagmentation buffer at 37°C for 30 min (Vazyme Biotech, TD502). Then, the stop buffer was added directly into the reaction to end the tagmentation. PCR was then performed to amplify the library for 15 cycles using the following PCR conditions: 72°C for 3 min; 98°C for 30 s. This was by thermocycling at 98°C for 15 s, 60°C for 30 s and 72°C for 3 min, followed by 5 min at 72°C. After the PCR reaction, libraries were purified with 1.2X AMPure (Backman, A63880) beads before proceeding to mitochondrial DNA depletion. A total of 400 sgRNAs targeting the mouse mitochondrial genome were provided as a gift by Wei Xie's laboratory of School of Life Sciences, Tsinghua University. Next, in vitro transcription was performed to produce sgRNAs using the MEGAshortscript? Kit (Ambion, AM1354)). Each ATAC-seq library was incubated with 5 μg of sgRNA and 10 μg of Cas9 protein (PNA Bio, CP02-50) for 2 h at 37°C. After incubation, the reaction was treated by RNase A before being terminated by adding stop buffer (30% glycerol, 1.2% SDS, 250mM EDTA, pH 8.0). The samples were then processed by an Illumina HiSeq 2500 sequencer with 50 bp paired-end sequencing reactions.
attributes.tag: GEO Accession
attributes.value: GSM2991101