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| Status |
Public on Aug 05, 2013 |
| Title |
OCI-LY1_siNT_24hrs_mRNAseq |
| Sample type |
SRA |
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| Source name |
Human DLBCL cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: DLBCL cell line OCI-LY1
genotype/variation: siNT mediated knockdown
time: 24hrs
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| Treatment protocol |
OCI-Ly1 cells were transfected using Nucleofector 96-well Shuttle system (Lonza) with siBCL6 (HSS100968) or siNT (46-2001) (Stealth RNAi, Invitrogen)
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| Growth protocol |
DLBCL cell lines OCI-Ly1 and OCI-Ly7 were grown in medium containing 90% Iscoveâs (Cellgro, Manassas, VA), 10% fetal bovine serum (Gemini, Irvine, CA), and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA).
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| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP-seq libraries were prepared using the Illumina ChIP-seq Library preparation Kit following the manufacture's instructions with minor modifications. Briefly 10ng of purified ChIP DNA was end repaired by conversion of overhangs to phosphorylated blunt ends. A' bases were added to the 3'ends of the DNA fragments and Illumina adapters (1:30 dilution) were ligated to the ends of ChIP fragments. After adapter ligation DNA was separated by electrophoresis and size selected by isolating a gel band of 250±25bp. This fragment range corresponds to a ChIP fragment range of about 158±25bp. Size selected fragments were PCR amplified for 15cycles using Illumina genomic DNA primers 1.1 and 1.2 with the following program (30s at 98oC, 15cycles of 10 at 98oC, 30s at 65oC,30s at 72oC and 5min extension at 75oC). Libraries were quantified and validated using Agilent Technologies 2100 Bioanalyser for size, concentration and purity. Q-PCR was repeated to confirm retention of relative enrichment.
RNAseq: Three ug of total RNA was isolated from OCI-Ly1 cells transfected using Nucleofector 96-well Shuttle system (Lonza) with siBCL6 (HSS100968) or siNT (46-2001) (Stealth RNAi, Invitrogen) at 24hrs and 48hrs after nucleofection. RNAeasy Plus Kit (Qiagen) that included a gDNA elimination step was used for RNA isolation. RNA concentration and purity were determined using Nanodrop (Thermo Scientific) and integrity was verified using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Libraries were generated using mRNA-seq sample prep kit by Illumina. Briefly, mRNA was selected by two rounds of purification using magnetic polydT beads and then fragmented. First strand synthesis was performed using random oligos and SupersciptIII (Invitrogen). After second strand synthesis a 200bp paired-end library was prepared following the Illumina paired-end library preparation protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
mRNA sequencing in OCI-Ly1 24hrs after siNT nucleofection
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| Data processing |
ChIP-seq: Raw images generated went through primary image analysis and basecalling (RTA v1.6) that was followed by Illumina Genome Analyzer Off-Line Basecaller (OLB v1.6) analysis, where reads were aligned to the human genome (UCSC hg18) using ELAND. Only sequences mapped uniquely to the genome with not more than 2 mismatches were used for downstream analysis. Several reads mapping to the same exact location (clonal reads) were considered amplification artifact and were excluded from the analysis. Table S2 includes information of the total number of mapped reads that where used in our analysis. Read density tracks were visualized using the UCSC browser.
ChIPseeqer peak detection algorithm (http://icb.med.cornell.edu/wiki/index.php/Elementolab/) was used for analyzing ChIP-seq data. Each read was in silico extended to 158bp based on the strand that it aligned to. Each ChIP-seq dataset was normalized to its corresponding input lane.
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mRNA-seq: Burrows-Wheeler alignment (BWA) algorithm was used to map paired reads using a 3-tiered hierarchal alignment first to a contaminant reference (containing adapter flanks, mitochondrial DNA and ribosomal DNA), then to the reference genome (human hg18), and finally to a junction database (which contains sequence which spans the boundaries between all concurrent exons as indicated by the annotation file). Gene expression values were calculated for each transcript (RPKM, reads per kilobase of exon per million mapped reads) by normalizing the number of bases aligned in a gene by the adjusted gene size (gene size minus any base that overlaps with other genes) and to the bases aligned to exons.
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| Submission date |
Jul 07, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Katerina Hatzi |
| E-mail(s) |
kac2029@med.cornell.edu
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| Organization name |
WCMC
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| Department |
Hematology/Oncology
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| Lab |
Ari Melnick
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| Street address |
413 E 69th Street, BB-1462
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE29282 |
A Hybrid Mechanism of Action for BCL6 in B Cells Defined by Formation of Functionally Distinct Complexes at Enhancers and Promoters |
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| Relations |
| SRA |
SRX084575 |
| BioSample |
SAMN00672580 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM756312_OCI-LY1_siNT_24hrs_mRNAseq.bed.gz |
708.4 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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