GSM3399568
GSM3399568: shTyr rep1; Homo sapiens; OTHER
RNA was extracted using TRIzol This procedure was conducted as described by (McGlincy and Ingolia, 2017). Briefly, cells were washed and flash frozen with liquid N2 before being lysed with lysis buffer containing cycloheximide (Alfa Aesar). Lysate was digested with RNase I (Lucigen) before ribosomes were isolated through ultracentrifugation through a sucrose cushion. The ribosome pellet was resuspended in a solubilization buffer containing 0.5% SDS and 1mM EDTA and TRIzol before RNA was extracted using the Direct-zol kit (Zymo Research). RNA was separated on a 15% TBE-Urea gel before RNA between 17nt and 34nt were gel extracted. Barcoded pre-adenylated linkers were ligated using T4 RNA ligase 2 truncated K227Q (NEB) and rRNA was depleted using the Ribo-Zero gold kit (Illumina) according to the manufacturer's protocol. RNA was converted to cDNA using SuperScript III (Life Technologies) and the RT product was circularized by CircLigase II (Epicentre). A PCR library was amplified and sequenced using Illumina Nextseq 500.
GEO
GSM3399568
GEO Accession
GSM3399568