GSM2817687: RPF_DHX9_1; Homo sapiens; OTHER
Total RNA and ribosome-protected fragments (RPFs) were isolated using the TruSeq Ribo Profile Mammalian Kit (Illumina) following the manufacturer's intructions with some modifications. For RPFs purification, lysates were treated with 5 U TruSeq Ribo Profile Nuclease per A260 of lysate. RPFs were isolated using Illustra MicroSpin S-400 HR Columns (GE Healthcare, 27- 5140-01). rRNAs were removed using the Ribo-zero Magnetic Gold kit (Illumina, MRZG12324). After PAGE purification (15% urea-polyacrylamide gel to select fragments ~28-30 nt in length), end-repair, 3'-adapter ligation, reverse transcription (EpiScript reverse transcriptase) and cDNA circularisation (CircLigase, Epicentre), RPFs were amplified using 9 PCR cycles using Phusion polymerase (NEB). For parallel RNA-seq, total RNA was processed similarly, but excluding the nuclease digestion and S-400 columns purification steps. Before the library preparation steps, total RNA samples were heat-fragmented (94°C for 25 minutes). PCR amplicons were purified on a 8% native polyacrylamide gel excising bands corresponding to ~70-80 nt and ~80-100 nt for RPF and total RNA samples respectively. Libraries were sequenced on a NextSeq 500 using 75-nt single-read sequencing runs.  For iCLIP, refer to associated publication. TruSeq Ribo Profile Mammalian Kit (Illumina) RNA-Seq, Ribo-Seq, iCLIP-seq
GEO Accession
GSM2817687