GSM6067628: Hg1; Homo sapiens; OTHER
RWPE-1 cells, grown to 80% confluence in supplemented K-SFM medium, were treated by adding formaldehyde directly to tissue culture medium to a final concentration of 1% and incubated for 10 minutes at room temperature. Cross-linking reactions were stopped by the addition of glycine to a final concentration of 125 mM. Cells were washed three times with ice-cold PBS, scraped, and centrifuged at 2,000 g for 5 minutes. Cell pellets were resuspended in lysis buffer freshly supplemented with protease inhibitor, PMSF and Superase-in (1ml lysis buffer/100 mg cell pellet) and immediately sonicated to an average chromatin length of 500-800 bp. Sonication was performed in a 4°C water bath at highest setting with 30 seconds ON, 45 seconds OFF pulse intervals. Sonicated samples were centrifuged at 16,100 g for 10 minutes at 4°C. 10 µl sonicated chromatin from each sample were stored as inputs for DNA extraction. Remaining chromatin was diluted in two times volume of hybridization buffer freshly supplemented with PMSF, proteinase inhibitor, and Superase-in. 100 pmol probes (LacZ-probes or MIR205HG-probes, sequences of all probes reported in PMID: 30659180) were added to 3 ml of diluted chromatin, which was mixed by end-to-end rotation at 37°C for 4 hours. Streptavidinmagnetic C1 beads (Invitrogen, Carlsbad, CA) were washed three times in lysis buffer and 100 µl washed C1 beads were added per 100 pmol of probes. The whole reaction was mixed for 30 min at 37°C. Beads:biotin-probes:RNA:chromatin adducts were captured by T-1-800 Adna Mag S magnets (AdnaGen, Langenhagen, Germany) and washed five times with wash buffer freshly supplemented with PMSF. After last wash buffer was removed carefully with P-10 pipette so that no trace volume was left behind. For DNA elution all samples including inputs were resuspended in DNA elution buffer and incubated at 37°C for 30 minutes with end-to-end rotation. Beads were captured by T-1-800 Adna Mag S magnets (AdnaGen, Langenhagen, Germany). Eluted DNA from two steps was combined. Chromatin was reverse crosslinked at 65°C overnight with 200 mM NaCl and treated with ProteinaseK at 45°C for 2 hours. DNA was then extracted with equal volume of phenol:chloroform:isoamyl (Invitrogen, Carlsbad, CA) and precipitated with ethanol at 80°C overnight. Isolated DNA was resuspended in elution buffer (19086, Qiagen, Hilden, Germany). High-throughput sequencing libraries from three independent ChIRP experiments were prepared and indexed using the ThruPLEX® DNA-seq Kit (Rubicon Genomics), using 12 cycles of PCR for all samples (MIR205HG and LacZ). Libriaries were purified on AMPure XP beads, then checked for quality and size range on Agilent Bioanalyzer using the High Sensitivity DNA Assay kit. Samples were then pooled and sequenced on Illumina HiSeq2000 with single read length of 100 bp (SR100).