title: GSM6276318: RNA-Klf5_KD-1; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq: Total RNA was extracted from cells by Trizol (Invitrogen, 15596-018), and 1 μg total RNA was reverse transcribed to obtain cDNA with cDNA Synthesis Kit (Vazyme, R212-01) according to the manufacturer's protocol.   ChIP-seq: For ChIP-seq, TSCs were crosslinked with 1% formaldehyde for 10 min, and quenched by 2 mL of 125 mM glycine for 5 min rotation at room temperature. Cells were washed 3 times with cold PBS. After centrifugation, cell pellets were lysed in buffer (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, pH 8.0, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 50 mM Tris-HCl, pH 8.0) for 10 min at 4℃. Chromatin was sonicated to 100-500 bp for the subsequent experiments.   ATAC-seq: ATAC-seq was performed as previously report[50]. In brief, a total of 50,000 cells were washed once with 50 µl of cold PBS and resuspended in 50 µl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). After centrifugation at 4℃, suspension of nuclei diluted to 5 ng/µl in the elution buffer (10 mM Tris buffer, pH 8) and followed by the addition of 50 µl transposition reaction mix of DNA Library Prep Kit V2 for Illumina (Vazyme, TD501). RNA-seq: Total RNA was extracted from TSCs by Trizol and RNA-Seq library were generated using RNA Library Prep Kit for Illumina® (NEB, E7530L) according to the manufacturer's recommendations.    ChIP-seq: The samples were pre-incubated with protein A/G Dynabeads (Life Technologies, 10015D) and the beads were then removed. Subsequently, the sample was added with 8 mg of Klf5(Sigma, 09-822) or H3K27ac antibody and rotated overnight at 4℃. Beads were washed four times with a Wash buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% glycerol, 0.5% Triton X-100, 0.01% SDS), eluted and reverse crosslinked. Paired-end 125 bp sequencing was performed on Illumina Hiseq X-Ten.   ATAC-seq: The reaction mix was gently mixed and incubated at 55℃ for 10 min. ATAC-seq library was amplified in 9 cycles. Libraries were purified using Qiagen PCR Cleanup Kit according to the manufacturer's instructions. Library concentrations were measured by KAPA Library Quantification Kit (KAPA Biosystems, KK4824) according to the manufacturer's instructions and checked by gel electrophoresis. Lastly, ATAC libraries were sequenced on Illumina Hiseq X-Ten.