GSM8757833: ATAC_sgKlf2_P14_Cl13_D5.5_3; Mus musculus; ATAC-seq
For ATAC-seq, 50,000 cells were pelleted at 500 g for 5 minutes and lysed on ice for 10 minutes in pre-chilled lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.01% Digitonin and 0.1% IGEPAL CA-630). Nuclei were harvested and subjected to a transposition reaction. For RNA-seq, 50,000 donor-derived P14 cells were sorted and lysed in lysis buffer (100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1.5% SDS) containing oligo-dT magnetic beads conjugated with Oligo-dT25. The mixture was incubated for five minutes to facilitate mRNA binding. Subsequently, the sample was placed on a magnetic rack to separate the beads, and the supernatant was discarded. The beads were washed twice with Wash Buffer A (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8; 0.1% SDS) and once with Wash Buffer B (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8) to remove non-specific contaminants. The supernatant was carefully removed after each wash. Elution of mRNA was achieved by adding 5 μL of Elution Buffer (10 mM Tris-HCl) and incubating the beads at 80°C for two minutes. The mixture was then subjected to magnetic separation, and the supernatant containing the extracted mRNA was collected. ATAC-seq was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501) with minor modifications.Nuclei were harvested to perform transposition reaction using TD501. The tagmented DNA was purified using the MinElute kit (Qiagen, 28004). Nextera indexing was performed on the tagmented DNA, which was amplified for 11 cycles as described, using a BIO-GENER GE4851T PCR machine. Amplified products were purified with VAHTS DNA Clean Beads, washed twice with 80% ethanol, and eluted in DEPC-treated Water. Libraries were sequenced on an Illumina NovaSeq platform, generating 150 bp paired-end reads.