GSM4976870: Rat Spg Chip-Seq_Sample 3; Rattus norvegicus; ChIP-Seq
Chip-Seq samples were extracted on d0 (prior to plating on laminin) or d7 post-plating on laminin (d5 in SD Medium), and ~4 million Brown Norway rat laminin-non-binding spermatogenic cells, using the RNAqueous Kit (Ambion, Inc.). RNA concentration in each isolated sample was measured using the RiboGreen Kit (Invitrogen, Inc.). Rat USg DNA (50 μg) was pretreated with protein G agarose beads (ThermoFisher) and Foxa2-DNA binding motifs co-precipitated using 5 μg of goat anti-human FOXA2 antibody sc-6554 (Santa Cruz Biotechnology) that was used to define the FOXA2 cistrome in the endometrium of the human uterus (Kelleher et al., 2019). Precipitates were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Cross-links were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation (Kelleher et al., 2019). Single end reads of 75 bp were generated. After mapping reads to rat genome Rnor6.0 by bowtie2 (v2.2.3) with parameter “--sensitive”, we perform filtering by (1) removing alignments with mapping quality less than 10, (2) removing duplicate reads identified by Picard MarkDuplicates (v1.127) http://broadinstitute.github.io/picard. Then enriched regions (peaks) were identified using MACS2 (v2.0.10)(Zhang et al., 2008), with a q-value cut-off of 0.05 for broad peaks. Peak regions were annotated by HOMER (Ross-Innes et al., 2012).