|
| Status |
Public on Aug 08, 2023 |
| Title |
CD4_HIVdelta6_SAHA_Pol2s2_CUTRUN_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
CD4_HIVdelta6
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: SAHA
replicate: Rep1
antibody: Pol2s2
method: CUT&RUN
|
| Treatment protocol |
As the reactivated cells returned to a resting state, cells were treated in triplicate with or without vorinostat (335nM) for 21d
|
| Growth protocol |
Healthy donor T cells were infected with HIV-dreGFP/Thy1.2 and isolated based on Thy1.2 surface expression. Infected cells were cultured for 21d, during which cells exhibited progressive decline in viral gene expression as latency is established. Latently infected cells were then re-stimulated with anti-CD3/anti-CD28 dynabeads to increase cell numbers and then allowed to return to latency
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
3x105 CD4 T cells per antibody condition were harvested, washed, and coupled to Concanavalin A-coated beads for 30 min at 4o C with mixing. Cells were incubated overnight in Digitonin-containing (0.01%) antibody buffer with the indicated antibodies at the following concentrations: IgG 1:50, H3K4me3 1:50, H3K27ac 1:100, H3K27me3 1:17, H3K9ac 1:17, H3K9me3 1:80, RNA pol2 1:17, RNApol2 P-S2 1:50. After incubation, cells were washed twice in cell permeabilization buffer (buffer CP, 0.01% Digitonin) and incubated with pAG-MNase for 15 min at room temperature. Cells were washed twice in buffer CP before CaCl2 was added for a 2h cutting reaction at 4oC with rotation. Stop buffer containing EDTA, RNase A, and E. coli spike-in DNA was added to halt the reaction during a 37° 10-min incubation step. Supernatants were collected and added to DNA binding buffer for subsequent DNA isolation by spin columns. Supernatant DNA yields were eluted from columns in 12μL of Tris-HCl elution buffer and quantified by Qubit High-Sensitivity dsDNA assay.
CUT&RUN Next-Generation Sequencing (NGS) library preparation was performed using NEBNext Ultra II DNA library preparation kits (New England Biolabs). DNA end repair and A-tailing was performed followed by adapter ligation to NEBNext hairpin adaptors and linearization of NEBNext adaptors with USER enzyme. Adaptor ligated libraries were amplified with sample-specific barcoded dual-index primers with PCR conditions optimized for amplification of short fragments: 14 cycles 15s at 98°C and 10s at 60°C. Libraries were quantified and examined by Qubit dsDNA assay (ThermoFisher) and capillary electrophoresis (Agilent Tapestation).
OTHER: CUT&RUN
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Data processing |
Reads were filtered for adapter contamination using Cutadapt96 and filtered such that at least 90% of bases of each read had a quality score>20.
Duplicated sequences were then capped at a maximum of five occurrences
Reads were aligned to a custom reference genome that concatenated GRCh38 (hg38) with the HIV-dreGFP/Thy1.2 sequence and E. coli K12 sequence each added as additional pseudo-chromosomes. Alignment was performed using STAR version 2.7.3a retaining only primary alignments. Reads overlapping regions of the genome deemed problematic by ENCODE were excluded
Regions of significant enrichment were identified using MACS2 with a threshold of q < 0.1, and H3K27me3 data were run in “broad” mode. A union set of peaks called for all samples and conditions was then assembled using BEDtools. Regions exhibiting significant changes in abundance between vorinostat and vehicle-control conditions were detected using CSAW wherein the tested windows were restricted to those overlapping the union peak set, and significance was reported if p < 0.01
Assembly: hg38-HIVp379d6-eColi_K12
Supplementary files format and content: bigWig files have been normalized to the total number of aligned reads, and eColi reads have been removed
|
| |
|
| Submission date |
Apr 01, 2022 |
| Last update date |
Aug 08, 2023 |
| Contact name |
Jeremy Simon |
| E-mail(s) |
jsimon@ds.dfci.harvard.edu, jeremy_simon@med.unc.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Department of Data Science
|
| Street address |
450 Brookline Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL30173 |
| Series (1) |
| GSE199946 |
A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV infected cells |
|
| Relations |
| BioSample |
SAMN27177254 |
| SRA |
SRX14711902 |
