|
| Status |
Public on Jan 19, 2016 |
| Title |
MDM Day 7 +UDG |
| Sample type |
SRA |
| |
|
| Source name |
MDM cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: --
cell type: Monocyte-Derived Macrophage (MDM)
agent: HIV NL4.3
|
| Treatment protocol |
MDM cells were either mock treated or infected with HIV NL4.3 and incubated for the number of days indicated in the sample name.
|
| Growth protocol |
Cells were harvested from peripheral blood of donors. PBMCs were from whole blood ficoll gradient and monocytes were isolated using the Pan monocyte Isolation Kit (Miltenyi Biotec). Monocytes were differentiated into macrophages over 7 d using MDM-20 media containing RPMI 1640, 20% (vol/vol) autologous plasma, 1 ng/mL GM-CSF (BD Biosciences), 1× Hepes, and 1× Glutamine (Gibco). Cultured MDMs were maintained in media containing RPMI 1640 + 10% (vol/vol) dialyzed FBS, 1% Pen/Strep.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using a Qiamp DNA extraction kit(Qiagen) according to manufacturer's protocol.
The isolated DNA was sheared for 12 minutes on a bioruptor with 30sec on 30 sec off. Sheared DNA was end repaired, a-tailed, and ligated to dual indexed long Illumina adapters. Libraries were mixed equimolar to 500ng and mixed with 5µg of cot1 and IDT blocking oligos and dried down. Lockdown probes against HIV in the forward and reverse direction were added in seperate reactions to 3pmol. Libraries were hybridized for 4 hours at 65˚ then bound to M-270 streptavin beads and washed. HIV-Lockdown DNA was eluted and samples were split and treated with or without 5U UDG and 10U EndoIV for 2 hours at 37˚. Samples were then PCRed with short Illumina primers for 22 cycles with Maxima Taq and purified with ampure beads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
JS215
HIV lockdown
|
| Data processing |
Reads were trimmed 2 bp on the 5' end for quality. Reads were aligned to the UCSC hg19 genome with HIV virus added as a chromosome using bwa mem. The command to run the alignment: bwa mem <bwaindex (hg19+HIV)> read1.fastq.gz read2.fastq.gz |samtools view -ShuF4 - | samtools sort -o - >bamfile
BAM alignment files were processed to genome coverage in bedGraph format using bedtools
BAM alignment files were processed to deduplicated bamfiles using samtools and piccard.
deduplicated bam alignment files were processed to vcf files using freebayes and vcffilter
BAM alignment files were processed to chimera and discordant alignments using pysam and UNIX
Segmentation analsysis was performed using chimeric and discordant reads and hepG1 segmentation data using bedtools
Genome_build: Hg19 +HIV(virus)
Supplementary_files_format_and_content: bedGraph files
Supplementary_files_format_and_content: VCF files: Contain variant calls (from FreeBayes) for the JS208 and JS215 samples and are produced using the pipeline at: https://github.com/hesselberthlab/stivers-hiv
Supplementary_files_format_and_content: Oligos.xlsx file: Contains all Illumina adaptor and PCR sequences, as well as Lockdown probes used for HIV isolation.
|
| |
|
| Submission date |
Dec 17, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jay R. Hesselberth |
| E-mail(s) |
jay.hesselberth@cuanschutz.edu
|
| Organization name |
University of Colorado School of Medicine
|
| Department |
Biochemistry and Molecular Gentetics
|
| Lab |
Jay Hesselberth
|
| Street address |
12801 E 17TH AVE
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE76091 |
Diverse fates of uracilated HIV DNA during infection of myeloid lineage cells |
|
| Relations |
| BioSample |
SAMN04348357 |
| SRA |
SRX1488161 |
