GSM3579927: ATACseq in Tpit Knockout intermediate pituitary rep1; Mus musculus; ATAC-seq
Mouse pituitary intermediate or anterior lobes were dissected pituitaries were separated into intermediate and anterior lobes and kept during the dissections in 300µl of dissection buffer (DMEM, 10% FBS, HEPES 10mM and DNase 100 U/ml). Anterior lobes were cut in pieces using a scalpel to facilitate tissue dissociation and digested at 37°C using 5mg/ml Trypsin for 10 minutes. We then added 2mM EDTA and incubated 5 minutes more. 50 000 cells were washed in PBS and incubated on ice for 30 minute in a hypotonic cell lysis buffer (0.1% w/v sodium citrate tribasic dehydrate and 0.1% v/v Triton X100) and centrifuged (5 minutes at 2000g at 4°C). Cells were then incubated 30 minutes on ice in cell lysis buffer (10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% v/v IGEPAL CA-630). After centrifugation, the nuclei pellets were resuspended in transposase Master Mix (1.25 µl 10x TD buffer, 5 µl H2O and 6.5 µl of Tn5: Illumina Nextera Kit; FC-121-1031) and incubated for 30 minutes at 37°C. Samples were purified using the DCC purification columns (Zymo). The eluted DNA was barcoded for multiplexing of samples using Nextera barcodes and PCR-enriched using the Phusion kit. Libraries were recovered with GeneRead Purification columns. Samples were then evaluated by qPCR to test enrichments and sequenced on Illumina Hiseq 2500 with 50bp or 125bp paired-end reads according to Illumina's recommendation.
GEO Accession
GSM3579927