GSM1572285: Aff3_Ab1_in_TC1_Zfp57_WT-rep1; Mus musculus; ChIP-Seq
For ChIP-Seq, 5×107 cells were used per immunoprecipitation according to previously described protocols (Lee et al., 2006). ChIP-Seq libraries were prepared with Illumina's kit or KAPA kit. For RNA-seq, cells were infected with lentivirus carrying either Non-targeting shRNA, Aff3 shRNA in the presence of 8 ug/ml of polybrene. 24 hours later, cells were selected with 2 ug/ml of puromycin for additional 48 hours and then ES were grown one passage off feeders for 30 min before harvesting. Total RNA was isolated with the RNeasy (74106 Qiagen) kit, treated with DNase I (M0303L NEB), and re-purified with RNeasy. For total RNA-seq analyses, total RNA was depleted of ribosomal RNA using Ribozero (Illumina) before library preparation performing Tru-seq sample prep (Illumina). For Nascent RNA-seq, about 1×108 fresh ES cells were washed twice in ice-cold PBS. The cells were then resuspended in Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT and proteinase inhibitors (Roche)) and incubated on ice for 15 min. Cell suspension was transferred to a pre-cold Dounce tissue grinder and homogenized 10 times by using a tight pestle on ice. After centrifugation the pellet containing enriched nuclei was washed twice in S1 buffer (0.25 M Sucrose, 10 mM MgCl2, 10 mM HEPES, pH 7.9, 1 mM DTT and proteinase inhibitors). To obtain the chromatin pellet, the nuclei pellet was then washed in ice-cold NUN buffer (20mM HEPES pH7.9, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP-40, 1 mM DTT, 20U/ml RNase Inhibitor (Ambion), and proteinase inhibitors) for 5 times. Chromatin bound RNA was isolated with RNeasy kit. The RNA was poly-A depleted by using Oligo(dT) magnetic beads (Invitrogen) and treated RNase free DNase I, and re-purified with the RNeasy column. 500 ng RNA was used for ribosomal RNA depletion with Ribo-zero Gold (Epicentre) and library preparation was performed with TruSeq Stranded Total RNA-seq Sample prep kit. ChIP-seq, total RNA-seq and nascent RNA-seq libraries were prepared for sequencing using standard Illumina or KAPA protocols
GEO
GSM1572285