GSM6025812: i2_HS30_S5_rep2; Homo sapiens; RIP-Seq
Chromatin isolation: Cells were transferred into a large volume of ice-cold PBS to instantly bring the temperature below 8°C. The cells were pelleted by centrifugation and washed with ice cold PBS. The cell pellets were resuspended in ice-cold NUN buffer and the samples vortexed and centrifuged at 12 500 G at 4°C for 30 minutes. The chromatin pellet was washed once in ice cold 50 mM Tris-HCl (pH 7.5) and stored at -80°C. PRO-IP-seq: Fragmented chromatin was subjected to a run-on reaction with biotinylated rA/rC/rG/rUTP. After the run-on reaction the samples was divided into distinct tubes for antibody-specific immunoprecipitation. After 3'-ligation of barcoded adapters (6nt barcode + constant G at the ligation site), the samples were re-pooled into a single tube. The nascet RNA was selected with streptavidin beads (x3 during the PRO-IP-seq protocol). The 5'-ends of nascent transcripts were decapped, repaired and ligated to UMI-containing adapters (6-nt UMI + constant C at the ligation site). The nascent RNA was reverse-transcribed, quality controlled and amplified to final sequencing library (Illumina TruSeq small RNA). Illumina NextSeq 500 and Illumina NovaSeq6000