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| Status |
Public on Sep 17, 2024 |
| Title |
J-Lat 10.6_ INTS12 KO_DMSO_R1 |
| Sample type |
SRA |
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| Source name |
Peripheral blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Peripheral blood
cell line: J-Lat 10.6
cell type: Jurkat
genotype: INTS12 knockout cells
treatment: DMSO
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| Treatment protocol |
J-Lat cells were knocked out for INTS12 or AAVS1 with single guides cloned in the HIV-CRISPR vector. Transduced cells were selected with 0.4 μg/mL puromycin selection for 10–14 days to generate CRISPR/Cas9-edited knockout pools. Knockout scores were generated by amplifying PCR products including the edited and non edited region and then using Synthego ICE analysis software to generate a knockout score. The knockout score of J-lats used in this experiment for INTS12 was 75%. AZD5582 (MedChemExpress, HY-12600) and I-BET151 (SelleckChem, S2780) were resuspended in DMSO to make 1 mM stocks stored at -80 °C. Stocks were then diluted down to single-use working concentrations for experiments with RPMI. AAVS1 KO and INTS12 KO cells were seeded at a density of 5e5 cells/ml in 96 well plates with 200 ul/well or in flasks. LRA treatments were performed in triplicate. Cells were treated with 10 nM AZD5582 & 100 nM I-BET151 for 48 hrs.
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| Growth protocol |
J-Lat 10.6 cells were cultured with RPMI 1640 media (ThermoFisher, 11875093) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin/Streptomycin, and 10 mM HEPES (ThermoFisher, 15630080).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed with 700 µl of QIAzol (Qiagen, 1023537). Samples were spun through a QIAshredder (Qiagen, 79654) and then frozen at -80°C. Cells were then processed with a miRNeasy kit (Qiagen, 217004), DNase treated with RNase-Free DNase Set (Qiagen, 79254), and then re-processed through the RNeasy Mini Kit (Qiagen, 74104) with the EtOH wash modified to 700 µL for small RNAs.
rRNA was removed with the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (20040525) followed by indexing with IDT for Illumina RNA UD Indexes Set A Ligation (20040553).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
A custom reference was built on the combined genome of human and HIV. HIV genome sequence (MN989412) was inserted into human chr9 at the known integrated site. 3' LTR of HIV was masked. GENCODE annotation v38 and HIV gene annotation were combined and genome coordinates were modified accordingly. STAR v2.7.7a with 2-pass mapping was used to align paired-end reads to the custom reference. FastQC 0.11.9, RNA-SeQC 2.3.4, RSeQC 4.0.0 were used to check various QC metrics including insert fragment size, read quality, read duplication rates, rRNA rates, gene body coverage and read distribution in different genomic regions. featureCounts in Subread 2.0.0 was used to quantify gene-level fragment counts in second-stranded fashion. Bioconductor package edgeR 3.36.0 was used to detect differential gene expression between sample groups. Genes with low expression were excluded using edgeR function filterByExpr with min.count = 10 and min.total.count = 15. The filtered expression matrix was normalized by TMM method and subject to significance testing using quasi-likelihood pipeline implemented in edgeR. A gene was deemed differentially expressed if absolute log2 fold change was above 1 (i.e. fold change > 2 in either direction) and Benjamini-Hochberg adjusted p-values was less than 0.05.
Assembly: hg38 with HIV sequence inserted on chr9.
Supplementary files format and content: A text file of gene-by-sample read counts. Gene symbols and sum of non-overlapping exon lengths are provided.
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| Submission date |
Sep 09, 2024 |
| Last update date |
Sep 17, 2024 |
| Contact name |
Michael Emerman |
| E-mail(s) |
memerman@fredhutch.org
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| Organization name |
Fred Hutch Cancer Research Center
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| Street address |
1110 Fairview Ave N
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE276747 |
Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [RNA-seq] |
| GSE277306 |
Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal |
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| Relations |
| BioSample |
SAMN43584856 |
| SRA |
SRX26058913 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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