GSM7832497: 01MLg_Mir9_ChIRP; Mus musculus; OTHER
Chromatin was diluted in two times volume of hybridization buffer (750mM NaCl, 1% SDS, 50mM Tris 7.0, 1mM EDTA, 15% formamide, add 1% of DTT, PMSF, PI, and 0.1% of RNase inhibitor (Promega). 150pmol of probes (Biotinylated tilling oligo of mature Mir9 or Mirctrl) added to ml of diluted chromatin, which was mixed by end-to-end rotation at 37°C for 4h. Streptavidin-magnetic C1 beads (Invitrogen) were washed three times in nuclear lysis buffer, blocked with 500ng/μl yeast total RNA, and 1mg/ml BSA for 1h at room temperature, and washed three times again in nuclear lysis buffer before resuspended in its original volume. 100µl of washed/blocked C1 beads were added per 100pmol of probes, and the whole reaction was mixed for another 30min at 37°C. beads:biotin-probes:RNA:chromatin adducts were captured by magnets (Invitrogen) and washed five times with40× beads volume of wash buffer (2× SSC, 0.5% SDS, add 1% DTT, PMSF and PI). After last wash buffer was removed carefully with P-10 pipette so that no trace volume was left behind. Beads are now poised for different elution protocols depending on downstream assays. Beads were resuspended in 3× original volume DNA elution buffer (50mM NaHCO3, 1% SDS, 200mM NaCl), and DNA was eluted with a cocktail of 100μg/ml RNase-A (Sigma-Aldrich). Chromatin was reverse crosslinked at 65°C overnight. DNA was then extracted purify the DNA with qiaquick columns according to manufacturer's instructions NEXseq