title: GSM4162177: THP-1 cells, Runx1_fRIP-seq; Homo sapiens; RIP-Seq
library_construction_protocol: fRIP was performed following a protocol reported by Hendrickson et al (Hendrickson et al., 2016) with modifications. Briefly, cells were crosslinked in 0.1% formaldehyde at room temperature for 10 minutes. The crosslinking reaction was quenched for 5 min at room temperature with 0.125 M glycine. Crosslinked cells were washed with ice-cold PBS. Cell pellet was lysed in RIPA lysis buffer (50 mM Tris (pH 8), 150 mM KCl, 0.1 % SDS, 1 % Triton-X, 5 mM EDTA, 0.5 % sodium deoxycholate, 0.5 mM DTT) supplemented with protease inhibitor cocktail (Thermo Scientific) and 100 U/ml RNaseOUT™ (Invitrogen). After sonication, cell lysate was pre-cleared by incubating with Dynabeads® Protein G (Invitrogen). Beads were then captured and removed using a magnet.  Pre-cleared lysate was incubated with anti-Runx1 antibody or IgG at 4°C for 2 h before adding 50 μl of Dynabeads® Protein G to capture antibodies. After washing, beads were kept at -200C or preceded to incubation with reverse-crosslinking buffer (3× PBS (without Mg or Ca), 6 % N-lauroyl sarcosine, 30 mM EDTA, 15 mM DTT) supplemented with Proteinase K (Ambion) and RNaseOUT together with input sample. Captured RNAs were extracted by Trizol reagent. Extracted RNA was treated with DNAse from RNase-Free DNase Set (QIAGEN) then ribosomal RNA was removed using the Ribo-Zero™ Magnetic Gold Kit (Epicentre). RNA quality was determined using the RNA 6000 Pico Kit on a Bioanalyzer (Agilent). cDNA libraries were constructed with the Truseq stranded total RNA library prep kit (Illumina) according to manufacturer's protocol. The libraries were pooled together and subjected to pair-end sequencing on a Nextseq500 (Illumina) to achieve 2×40 bp reads.
attributes.tag: GEO Accession
attributes.value: GSM4162177