alias: GSM4717233
title: GSM4717233: WT_Cytosolic; Homo sapiens; RNA-Seq
library_construction_protocol: Approximately 2-5 million HEK293T wild type or mutant cells were collected and the RNA was extracted using the Zymo RNA miniprep kit following the manufacture's manual. Genomic DNA was removed by on-column digestion of DNase I. Human embryonic stem cells (hESCs) and the differentiated neural progenitor cells (NPCs) were collected and the RNA was extracted using the Trizol method. Briefly, the cells were resuspended in 500ul-1000ul Trizol and incubated at room temperature for 5 minutes and debris was removed after centrifugation. 0.2 volume of chloroform was added to the supernatant. Aqueous phase was collected after centrifugation at 4C and mixed with 1 volume of isopropanol at -20C for more than 30 minutes. RNA was precipitated at 13,000 rpm 4C. 1-2ug RNA was used for cDNA synthesis using the iScript Advanced kit (Bio-Rad) following the manual. rRNA was depleted from total RNA using RNase H-based protocols adopted from (Adiconis et al., 2013; Morlan et al., 2012). In brief, approximately 100-1000 ng of RNA was annealed with pooled rRNA antisense DNA oligo (gift from J. Salzman lab at Stanford) by heating at 94°C  and slowly cooling down to 45°C. Hybridase Thermostable RNase H (Epicenter, Madison, WI: Lucigen H39500) was added to the mix and incubated at 45°C on a thermocycler for 30 minutes to remove rRNA. rRNA-depleted RNA was then purified using Agencourt RNAClean XP beads (Beckman Coulter: A63987). Excessive DNA oligo was removed by treatment of TURBO DNase (Invitrogen: AM1907). The RNA was then purified with RNAClean XP beads again and was used as RNAseq library prep input using the KAPA HyperPrep RNA-seq Kit (Kapa Biosystems: KK8540).
identifiers.namespace: GEO
identifiers.id: GSM4717233
attributes.tag: GEO Accession
attributes.value: GSM4717233