title: GSM2938890: ChIP_INPUT_Mll2CD; Mus musculus; ChIP-Seq
library_construction_protocol: ChIPseq: ChIP experiments were carried out following a standard protocol in the lab (Morey et al, Cell Stem Cell 2013). PROseq: We followed the protocol as described previously (Mahat et al, Nat Protoc., 2016) with some modifications. After 4-hydroxytamoxifen treatment for 96 h, nuclei were isolated by Dounce homogenizer with loose pestle. ~10^7 nuclei were subjected to nuclear run-on (30 °C, 3 min) in the presence of 25 µM Biotin-11-ATP/GTP/CTP/UTP (PerkinElmer) and 6x10^5 Drosophila S2 spike-in nuclei. Total RNA were extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNA were purified by Dynabeads M-280 streptavidin (Invitrogen). After 3' VRA3 adapter ligation and the 2nd purification by Dynabeads, 5' cap and 5' hydroxyl RNA were converted to 5' monophosphorylated RNA by RppH (NEB) and PNK (NEB), respectively. After 5' VRA5 adapter ligation and the 3rd purification by Dynabeads, cDNA were generated by reverse transcription with SuperScript III (Invitrogen) and RP1 primer. PCR by Phusion Hot Start (Thermo) and RP1/RPIx primer sets in 10 cycles amplified indexed DNA libraries. 140–350 bp DNA libraries were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science) and then sequenced by NextSeq 500 system (illumina) with single-read runs. Libraries were prepared according to Illumina instructions.
attributes.tag: GEO Accession
attributes.value: GSM2938890