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| Status |
Public on Feb 26, 2013 |
| Title |
LIN28B_knockdown_rep2 |
| Sample type |
SRA |
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| Source name |
HEK 293 cells inducibly expressing FLAG/HA tagged LIN28B; transgene not induced; siRNA transfected; replicate 2
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
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| Treatment protocol |
Expressoin of LIN28B was induced with 0.25 µg/ml of doxycyclin
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| Growth protocol |
HEK293 T-REx Flp-In cells (Invitrogen) were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin, 100 µg/ml zeocin and 15 µg/ml blasticidin. Cell lines stably expressing FLAG/HA-tagged LIN28B were generated by co-transfection of pFRT/TO/FLAG/HA/LIN28B construct with pOG44 (Invitrogen). Cells were selected by exchanging zeocin with 100 µg/ml hygromycin. Expression of FLAG/HA-LIN28B was induced by addition of 250 ng/ml doxycycline 15 to 20 h before crosslinking.
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| Extracted molecule |
total RNA |
| Extraction protocol |
TRizol extraction according to the manufacturer's protocol
small RNA cDNA library according to Hafner et al, Methods, 2012, and Hafner et al., RNA, 2011
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Gene expression data from HEK293 cells inducibly expressing FLAG/HA tagged LIN28B after knockdown with siRNA
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| Data processing |
Reads are assigned to their respective samples using custom perl scripts. Requirements are that the barcode in its entirety must be found, and the first 5 nt of the 3' adapter sequence allowing for one error (mismatch, indel). Reads ranging from 19-15 nt are analyzed
Mapping against the human genome release hg19 and custom non-coding (ncRNA) RNA database is performed using BWA version 0.5.9, with paramters "-n 2 -t 12" against the ncRNA database, "-n 1 -t 12" against the human genome
Cutsom scripts are used to assign reads first by assigning reads by lowest error maping error, then by hierarchy accounting for relative biological abundance of ncRNA species as well as enrichment of 5'-p 3'-OH small RNA cDNA cloned according to protocol outlined above.
Genome_build: hg19, custom annotation database containing genbank defined ncRNAs and in-house miRNA definitions
Supplementary_files_format_and_content: first column contains the read with barcode and adapter removed, the second the read count, the third the annotated miRNA name or non-coding RNA type if applicable
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| Submission date |
Feb 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Miguel Brown |
| E-mail(s) |
miguel.a.brown@gmail.com
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| Organization name |
The Rockefeller University
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| Street address |
1230 York Ave Box 186
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE44568 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition [RNA-Seq] |
| GSE44616 |
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition |
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| Relations |
| SRA |
SRX245352 |
| BioSample |
SAMN01924687 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1087028_LIN28B_knockdown_rep2.txt.gz |
11.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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