GSM2521599: SSB PAR-CLIP, RNase T1; Homo sapiens; OTHER
Total RNA for sequencing was isolated using the TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Crosslinked RNA was recovered as described previously (Garzia et al. 2016), with the exception that only the first RNase step was performed. In one of the two replicates (SSB_RNase_A), RNase T1 was replaced by RNase A. For hydro-tRNAseq: 20 μg of 60-100 nt-sized total RNA were hydrolyzed in a 15 μL buffer of 10 mM in a 15 µL buffer of 10 mM Na2CO3 and 10 mM NaHCO3 at pH 9.7 either at 65 °C for 10 min (replicate 1) or 1 h (replicates 2-4). The partially hydrolyzed RNA was dephosphorylated with 10 U of calf intestinal phosphatase (NEB) in a 50 µL reaction of 100 mM NaCl, 50 mM Tris-HCl, pH 7.9 at 25 °C, 10 mM MgCl2, 1 mM DTT, 3 mM Na2CO3 and 3 mM NaHCO3, at 37 ºC for 1 h. The resulting RNA was re-phosphorylated with 10 U of T4 polynucleotide kinase (NEB) in a 20 µlL reaction of 70 mM Tris-HCl, pH 7.6, 10 mM MgCl­2, 5 mM DTT and 1 mM ATP, at 37 °C for 1 h. Fragments of 19-35 nt were converted into barcoded small RNA cDNA libraries. Replicate 1 was run alone on a single lane with 3' adapter:TCGTATGCCGTCTTCTGCTTG. Replicates 2-4 were part of a barcoded library. 3' adapters used (5 first nts represent barcodes): replicate 2, TCACTTCGTATGCCGTCTTCTGCTTG; replicate 3, TCACTTCGTATGCCGTCTTCTGCTTG; replicate 4, TCTAGTCGTATGCCGTCTTCTGCTTG. The recovered RNA was carried through a cDNA library preparation protocol as described previously (Garzia et al. 2016).