GSM6509637: Leptonema/Zygonema ChRO-seq rep 1; Mus musculus; OTHER
Prophase I substages were isolated using STAPUT. 10 million cells were used as input to extra total chromatin. Briefly, chromatin from enriched prophase I substages was mixed with 50 µl 2x run-on master mix (10 mM Tris-HCl pH 8.0, 5 mM MgCl2,1 mM DTT, 300 mM KCl, 10 mM Biotin-11-CTP (Perkin Elmer # NEL542001EA), 400 uM ATP (NEB N0450S), 0.8 uM CTP (NEB N0450S), 400 uM GTP (NEB N0450S), 400 uM UTP (NEB N0450S), 40 uM Biotin-11-CTP (Perkin Elmer # NEL542001EA), 40 units SUPERase in RNase Inhibitor (Life Technologies AM2694), and 1% Sarkosyl (Fischer Scientific #AC612075000). The run-on reaction was incubated at 37˚C for 5 minutes and then stopped by adding Trizol LS (Life Technologies 10296010). RNA was extracted by adding chloroform and precipitating the aqueous phase with GlycoBlue and ethanol. Unincorporated biotin nucleotides were removed with Micro Bio-Spin P-30 Gel Columns, Tris Buffer (RNase-free)  (Bio-Rad #7326250). Nascent RNA was purified by binding streptavidin beads (NEB S1421S) and washed as described by Mahat et al., 201663. The RNA was removed from beads by Trizol and then followed with the 3' adapter ligation using T4 RNA Ligase 1 (NEB M0204L). After this, a second bead binding was performed followed by a 5' de-capping using RppH (NEB M0356S). The 5' end of the transcript was phosphorylated using PNK (NEB M0201L) and then purified with Trizol (Life Technologies 15596-026). After this, a 5' adapter was ligated onto the RNA transcript using T4 RNA Ligase I (NEB M0204L), and a third bead binding was performed. RNA was purified again with a Trizol and ethanol extraction A reverse transcription reaction was performed in order to generate cDNA using SuperScript IV Reverse Transcriptase (Life Technologies 18090010). The cDNA was amplified with Q5 High-Fidelity DNA Polymerase (NEB M0491L) in order to generate the leChRO-seq libraries which were prepared using manufacturer's protocol (Illumina) and single-end sequenced by means of Illumina NextSeq500 at Cornell University Biotechnology Resource Center.