|
| Status |
Public on Oct 28, 2013 |
| Title |
RNA-Seq_CapH2_R1 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: V6.5 embryonic stem cells
cell count: 6.52E+05
shRNA: TRCN0000175785
barcode (already removed): CGTACG
|
| Treatment protocol |
shRNA plasmids targeting the mouse CapH2, Smc2, Smc1 mRNA and GFP (control) (Open Biosystems, Huntsville) were used. Sample CapH2 R1(also known as #3) used hairpin TRCN0000175785, CapH2 R2 (also known as #4) used TRCN0000175531, Smc2 R1 also known as #1) used TRCN0000108952, Smc2R2 (also known as #2) used TRCN0000108953, Smc1 used TRCN0000109033, and GFP used TRCN0000072201. Viral media was collected 48 hours after co-transfection and the V6.5 mES cells were directly infected with the viral media 24 hours after initial plating of the mES cells. The infection media was 1:5 viral media:mES cell media with 8ug/ml polybrene. The efficiently infected cells were grown for 24 hours before selection with 0.5ug/ml puromycin for 48hours, then 1ug/ml puromycin for 48 more hours. V6.5 cells were cross-linked 96 hours post selection and frozen for experiments such as RNA-Seq, measuring knockdown efficiency and cell counting.
|
| Growth protocol |
V6.5 (C57BL/6-129) murine ES cells were grown under typical ES conditions on irradiated mouse embryonic fibroblasts (iMEFs). For expression analysis, cells were grown for one passage off of iMEFs, on gelatinized tissue-culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was performed using the miRVana miRNA extraction kit (Ambion, AM1560) and the recovered RNA was eluted in 30 μl elution buffer. Cell numbers were counted from proxy plates using C-Chip disposable hemocytometers (Digital Bio). An amount of RNA that corresponds to 6.52E+05 cells was used for subsequent steps. Total RNA concentrations were measured using the NanoDrop ND-1000. Synthetic RNAs (ERCC ExFold RNA Spike-In kit, Ambion, 4456739) were added to each sample based on cell number. For each sample 1.42 ul of a 1:10 dilution of the synthetic RNA Mix 1 was added to each RNA sample.
Libraries were prepared using the TruSeq RNA Kit version 2 (Illumina, RS-122-2001) according to manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-Seq from mES cells after knockdown of CapH2.
|
| Data processing |
Images analysis and base calling was done using the solexa pipeline.
For all RNA-Seq samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 1 -m 2 -n 2 --best --sam. Seed length (-l) was set to read length for each dataset.
Genome_build: mm9
Supplementary_files_format_and_content: RNA-Seq RPKM expression values were computed for each gene using RPKM_count.py 2.3.5.
|
| |
|
| Submission date |
Apr 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard A Young |
| E-mail(s) |
young_computation@wi.mit.edu
|
| Phone |
617-258-5219
|
| Organization name |
Whitehead Institute for Biomedical Research
|
| Lab |
Young Lab
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE46316 |
Transcription-dependent positioning of Structural Maintenance of Chromosome complexes across the genome: RNA-Seq |
|
| Relations |
| BioSample |
SAMN02055474 |
| SRA |
SRX271424 |
