|
| Status |
Public on Jul 25, 2013 |
| Title |
HBG CRISPRs, Rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T cells transiently transfected with Cas9 and HBG1/2 guide RNA expressing plasmids
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
guide rna: HBG1,HBG2
replicate: 3
|
| Treatment protocol |
For IL1RN-CRISPRs and HBG-CRISPRs, each well was transfected with 3 ug of Cas9-expressing plasmid, 250 ng (each) of four different guide RNA-expression PCDNA vectors, and 10 uL of lipofectamine in a total volume of 500 uL. For control experiments, 4 ug of empty PCDNA vector was transfected instead. RNA was harvested after three days.
|
| Growth protocol |
HEK293T cells were grown in DMEM with 10% fetal bovine serum and 1% Penicillin/Streptomycin in 6-well cell culture plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Qiagen Mini RNA prep kits. PolyA+ RNA was subsequently purified using oligo-dT beads (Invitrogen).
First strand cDNA was generated using SuperScript Vilo cDNA synthesis master mix (Invitrogen), and second strand cDNA was synthesized using E. coli DNA Polymerase I (New England Biolabs) with random hexamer primers. Double stranded cDNA was purified using 1.8X Agentcourt SPRI beads (Beckman Coulter). Double stranded cDNA was fragmented and sequencing adaptors added using Nextera transposase (Illumina) and indexed for sequencing by PCR as described previously. Samples were pooled in equimolar ratios and sequenced on an Illumina HiSeq 2000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
HBG.Rep3
PolyA RNA
|
| Data processing |
Base-calling was performed on instrument using CASAVA software.
Reads were aligned to known RefSeq transcripts from hg19 using Bowtie version 0.12.9, reporting only the best alignment by using the --best option.
Read counts were normalized and differential expression was called using Deseq (v. 1.10.1) by using a chi-squared test to compare fits to a model that contained donor and count (i.e. count ~ donor + condition) to a model that only contained donor information (i.e. count ~ donor).
P-values were converted to a False Discovery Rate using the method of Benjamini and Hochberg (1995).
Genome_build: RefSeq transcripts from genome build GRCh37
Supplementary_files_format_and_content: GSE47114_All_counts.tab: Tab-delimited matrix; Raw (not normalized) counts of the number of reads aligned to each RefSeq transcript.
|
| |
|
| Submission date |
May 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy E Reddy |
| E-mail(s) |
tim.reddy@duke.edu
|
| Organization name |
Duke University
|
| Department |
Department of Biostatistics & Bioinformatics
|
| Lab |
ReddyLab
|
| Street address |
2347 CIEMAS, 101 Science Drive
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE47114 |
RNA-Guided Human Gene Activation by Cas9/CRISPR-Based Engineered Transcription Factors |
|
| Relations |
| BioSample |
SAMN02147232 |
| SRA |
SRX283662 |
