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Sample GSM860025 Query DataSets for GSM860025
Status Public on Feb 20, 2012
Title hnRNPU 293T RNAseq KD2b
Sample type SRA
 
Source name human 293T cells
Organism Homo sapiens
Characteristics treatment: hnRNPU siRNAs
cell line: 293T
read length: 50
Treatment protocol Cells were transfected at 40% confluency using 5μl/ml Lipofectamine-2000 (Invitrogen) and siRNAs at a final concentration of 100nM. For hnRNP A1, A2/B1, H1, M and U, equal mixtures of multiple siRNA sequences were used and for hnRNP F only one siRNA sequence was used. Media was changed 4-6 hrs after the initial transfection, and the transfection was repeated 48 hrs later. Cells intended for RNA analysis were suspended and lysed in TRIzol (Invitrogen) 72 hrs after the first transfection.
Growth protocol Human 293T cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS at 37 ̊C and 5% CO2.
Extracted molecule polyA RNA
Extraction protocol Strand-specific RNA-seq libraries were prepared based on the method described in Parkhomchuk et al., 2009, with a few modifications. 10 μg of total RNA was DNase treated (Turbo DNase, Ambion) and then subjected to 2 rounds of polyadenylation selection using Oligo (dT)25 Dynabeads (Invitrogen) according to manufacturer’s recommendations. Eluted mRNA was then reverse transcribed with a mixture of random hexamers and oligo- dT primers. Unincorporated nucleotides were then removed using G-25 spin columns (GE Healthcare) and the second strand of the cDNA was then synthesized in the presence of dUTP instead of dTTP. This reaction was then brought to 350 μl and sonicated on high for 1 hour of 30 seconds on, 30 seconds off sonication bursts. Fragments ranging from 150-225 bp were then selected from a 6% polyacrylamide gel, before the ends of each fragment were repaired using T4 DNA Polymerase, Klenow DNA Polymerase, and T4 PNK (NEB) at 20°C for 1 hour. Enzymes, buffers, and nucleotides in all reactions were removed using Qiagen PCR Purification MinElute columns. Following the end repair, a single dATP was added to each 3′ end using Klenow Exo minus (NEB) at 37°C for 1 hr. The adaptors were then ligated using 2000 units per μl T4 DNA ligase (NEB) at room temperature for 30 minutes. The dUTPs were then removed using Uracil N-Glycosylase (Fermentas) at 37°C for 30 minutes. One half of the library was then subjected to 15 cycles of PCR amplification using Phusion High Fidelity DNA Polymerase (NEB). The library was then separated on a 6% polyacrylamide gel and fragments between 250-325 nucleotides were selected. 8 pM of the resulting amplified libraries were subjected to standard Illumina sequencing protocols.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description strand-specific RNAseq, hg18
Data processing Strand-specific RNA-seq reads from control siRNA treatment, and each hnRNP depletion experiment were processed as previously described (Polymenidou et al., 2011). An average of 70% of reads mapped uniquely to our gene structure database, using Bowtie (version 0.12.2, with parameters –l 20 –m 5 –k 5 ––best ––un –– max –q).
 
Submission date Jan 10, 2012
Last update date May 15, 2019
Contact name Gene Yeo
E-mail(s) geneyeo@ucsd.edu
Organization name UCSD
Street address 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
 
Platform ID GPL11154
Series (2)
GSE34995 Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins (RNA-Seq)
GSE34996 Integrative genome-wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins
Relations
SRA SRX115539
BioSample SAMN00771658

Supplementary file Size Download File type/resource
GSM860025_hnRNPU_293T_RNAseq_KD2b.hg18.bowtie.gz 330.4 Mb (ftp)(http) BOWTIE
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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