GSM4089240: HEK293_rep1_hydroseq; Homo sapiens; ncRNA-Seq
Hydro-tRNAseq: Total RNA was resolved on 15% Novex TBE urea gels and size-selected for 60-100 nt fragments. The recovered material was then alkaline hydrolyzed (10mM sodium carbonate and 10mM sodium bicarbonate) for 10 minutes at 60ºC. The resulting RNA was de-phosphorylated with Antarctic Phosphatase (New England Biolabs) at 37ºC for 1 hour. De-phosphorylated RNA was purified with an RNeasy MinElute spin column and re-phosphorylated with Polynucleotide Kinase (NEB). PNK-treated tRNAs were purified with an RNeasy MinElute spin column and, similar to small RNA-seq library preparation, adaptor-ligated, reverse-transcribed and PCR-amplified for 14 cycles. Cells were grown in 60mm dishes for 48h. Total RNA from HeLa, HEK293, HCT116, MDA-MB-231 and fibroblast BJ/hTERT was extracted using the miRNeasy Mini kit. Independent replicates where grown and RNA was extracted on different days. Upon applying either the hydro-tRNAseq or small RNA-seq protocol, the resulting cDNA was purified using a QIAQuick PCR Purification Kit. The adaptor ligated to the 3' end of the RNA is the standard TruSeq adaptor: 5'-AGATCGGAAGAGCACACGTCT-3'. The adaptor ligated to the 5' end is the TruSeq smallRNA adaptor: 5'-GTTCAGAGTTCTACAGTCCGACGATC-3'.
GEO Accession
GSM4089240