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| Status |
Public on Feb 20, 2012 |
| Title |
hnRNPU 293T RNAseq KD2b |
| Sample type |
SRA |
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| Source name |
human 293T cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: hnRNPU siRNAs
cell line: 293T
read length: 50
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| 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.
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| Growth protocol |
Human 293T cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS at 37 ̊C and 5% CO2.
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
strand-specific RNAseq, hg18
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| 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).
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| Submission date |
Jan 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
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| Organization name |
UCSD
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| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| 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 |
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| 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 Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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