A critical subpopulation of T-lymphocytes involved in the induction of most immunological functions. The HIV virus has selective tropism for the T4 cell which expresses the CD4 phenotypic marker, a receptor for HIV. In fact, the key element in the profound immunosuppression seen in HIV infection is the depletion of this subset of T-lymphocytes.
Attributes by Original Data Submitter
Naive CD4 T-cells
days after activation
Metadata from Sequence Read Archive
3 x 107 cells were fixed for 10 min in cell culture medium with 1% formaldehyde at room temperature. Fixation was quenched with 10% glycine for 5 min at room temperature. Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until chromatin isolation. Chromatin was isolated from cell pellets using the ChIP-It Express Enzymatic Shearing Kit (Active Motif, #53035) per the manufacturer’s instructions (www.activemotif.com). For immunoprecipitation, 7.5 μg of chromatin was diluted in a total of 1 mL of low salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl) with protease inhibitor cocktail and pre-cleared with 30 μL of Dynal Protein G magnetic beads (Invitrogen, #10004D) for 2 h at room temperature with rotation. 20 μL of pre-cleared chromatin was saved for input analysis and stored at -80°C. Remaining chromatin was incubated with 2 μL of anti-H3K4me2 antibody (Millipore, #07-030) or 4 μL of anti-H3K4me3 antibody (Millipore, #07-473) overnight at 4°C with rotation. 30 μL of Dynal Protein G beads were added to each ChIP and incubated at 4°C with rotation for 2 h. Beads were washed three times in low salt wash buffer and then 2 times with high salt wash buffer (0.1% SDS, 1.0% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl) with 5 min of rotation at 4°C for each wash. Beads were resuspended in 150 μL of elution buffer (1% SDS, 0.1M NaHCO3) and incubated in a thermomixer at 65°C for 30 minutes at 1,200 rpm to reverse cross-linking. 2 μL of Proteinase K (Invitrogen, #AM2546) was added to each sample, and 6 μL of 5 M NaCl was added for a total concentration of 200 mM. Samples were then incubated in a thermomixer at 65°C overnight at 1,200 rpm. Eluted samples were removed from the beads and purified using the Qiaquick PCR purification kit (QIAGEN, #28104) per the manufacturer’s instructions (www.qiagen.com). For ChIP-Seq, 10 ng of purified DNA from individual chromatin IPs were end repaired and A-tailed. Indexed adapters were ligated, and ligation product was purified on Agencourt Ampure XP beads and processed followed by size selection from 2% agarose. Purified product was amplified with 15 cycles of PCR followed by size selection from 2% agarose. Libraries were assessed on an Agilent Bioanalyzer using a DNA chip and quantitated using the Quant-iT ds DNA BR Assay kit (Invitrogen, #Q32853) and a Qubit Fluorimeter (Invitrogen). Cluster generation and sequencing on an Illumina HiSeq system was conducted directly with purified libraries following manufacturer’s instructions (www.illumina.com). 100 bp single-end reads were generated for naïve and memory CD4 T cells from 4 donors with 2 samples per lane.