|
| Status |
Public on Nov 15, 2018 |
| Title |
THP_BG505_viralRNA_2 |
| Sample type |
SRA |
| |
|
| Source name |
Human Immunodeficiency Virus (HIV)
|
| Organism |
Human immunodeficiency virus |
| Characteristics |
cell type: ZAP-KO THP-1 +CCR5
genotype: ZAP-KO
ifn treatment: yes
|
| Treatment protocol |
8e6 cells (>500X coverage) were transduced with the PIKAHIV library (MOI of 0.75) and selected in 1ug/mL puromycin for 10-14 days. For each replicate, 8e6 cells were infected with or without overnight IFNα treatment with HIV. Cells and viral supernatants were harvested 3 days post-infection.
|
| Growth protocol |
THP-1 monocytic cells are grown in RPMI with 10% FBS and Pen/Strep (supplemented to 10mM HEPES, 4.5 g/L D-Glucose, 0.11 g/L Sodium Pyruvate + Glutamax), passaging between 2e5 cells/mL and 1e6 cells/mL.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen)
Genomic DNA or reverse-transcribed viral RNA were amplified by a two-step PCR (PCR1: 12 cycles, PCR2: 20 cycles) to amplify 20bp sgRNA sequences in the HIV-CRISPR vector. All Viral RNA was amplified for each sample where appropriate. For Genomic DNA samples a total of 500-fold coverage of the 15,414 sgRNAs were amplified (10^6 cells were estimated to contain 6.6μg gDNA) with a maximum of 2μg gDNA per PCR reaction. For each sample, PCR1 reactions were pooled, cleaned up over a QIAquick PCR purification Kit (2 columns per sample), pooled again and used as template for PCR2. For viral RNA, all PCR1 was amplified in the second round. For gDNA, 5uL of pooled PCR1 was used as input for PCR2 (PCR2 primers include Illumina barcodes and adaptor sequences). Amplicons from PCR2 were pooled, purified by double-sided SPRI (AMPureXP, Beckman Coulter), quantified with a Qubit HS dsDNA assay and pooled for sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
viral RNA
|
| Data processing |
Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18 software, followed by generation of fastq files using Illumina's bcl2fastq Conversion Software v1.8.4 (http://support.illumina.com/downloads) /bcl2fastq_conversion_software_184.html).
Reads of low quality were filtered out prior to demultiplexing using FASTX-Toolkit v0.0.13
Reads were trimmed and aligned to the PIKA HIV guide library using Bowtie v1.0.0, allowing 0 mismatches in the guide sequence, followed by tabulation of read counts using R
Genome_build: PIKA HIV sgRNA Library
Supplementary_files_format_and_content: tab delimited files with read counts for each guide sequence
|
| |
|
| Submission date |
Nov 14, 2018 |
| Last update date |
Nov 15, 2018 |
| Contact name |
Molly Ohainle |
| E-mail(s) |
mohainle@fredhutch.org
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Street address |
1100 Fairview Ave N, C2-023
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL25457 |
| Series (1) |
| GSE118631 |
A Virus-Packageable CRISPR Screen Identifies Host Factors Mediating Interferon Inhibition of HIV |
|
| Relations |
| BioSample |
SAMN10424684 |
| SRA |
SRX5008748 |
