GSM1644021: ATAC-Seq from v6.5 embryonic stem cells; Mus musculus; OTHER
Nanog-CreER reporter MEFs were reprogrammed using OSKM-TD protocol until the end of OSKM induction step, at which point tdTomato+ intermediate cells were sorted and applied for ATAC sequencing. Briefly, 50,000 sorted cells were centrifuged at 500g for 3 minutes, followed by a wash using 50 μL of cold PBS and centrifugation at 500g for 3 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min using a refrigerated centrifuge. Next, the pellet was re-suspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase (Illumina) and 22.5 μL nuclease-free water). The transposition reaction was carried out for 30 minutes at 37 °C and immediately put on ice. Directly afterwards, the sample was purified using a Qiagen MinElute kit. Following purification, the library fragments were amplified using custom Nextera PCR primers 1 and 2 for a total of 12 cycles. Following PCR amplification the libraries were purified using a Qiagen MinElute Kit.