ChIPmentation of CD8+ T Cell Subsets

1 × 10⁶ FACS-sorted stem-like (Blimp-1-YFP^loTim3^lo), terminally exhausted (Blimp-1-YFP^hiTim3^hi), memory precursor (KLRG1^lo), and short-lived effector (KLRG1^hi) P14 cells were used for ChIP analyses. More than ten mice from each infection were pooled for each sort. Two biological replicates of each CD8⁺ T cell population were collected. DNA-protein crosslinking, nuclei isolation, and chromatin sonication were performed using truChIP Chromatin Shearing Kit (Covaris) according to manufacturer’s instructions. After immunoprecipitation by anti-H3K27Ac (ab4729, AbCam), ChIPmentation was performed according to the published protocol^{50 (link)}. The libraries were sequenced for 50 cycles (single read) on a HiSeq 3000 (Illumina).

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Yao C., Sun H.W., Lacey N.E., Ji Y., Moseman E.A., Shih H.Y., Heuston E.F., Kirby M., Anderson S., Cheng J., Khan O., Handon R., Reilley J., Fioravanti J., Hu J., Gossa S., Wherry E.J., Gattinoni L., McGavern D.B., O’Shea J.J., Schwartzberg P.L, & Wu T. (2019). Single-Cell RNA-Seq Reveals TOX as a Key Regulator of CD8+ T cell Persistence in Chronic Infection. Nature immunology, 20(7), 890-901.

Publication 2019

truChIP Chromatin Shearing Kit ab4729 HiSeq 3000

Biological Blimp 1 Cd8 t cell Cells Chip Chromatin Immunoprecipitation Infection Isolation Memory Mice Nuclei Protein Stem

Corresponding Organization :

Other organizations : National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Cancer Institute, National Institute of Neurological Disorders and Stroke, National Human Genome Research Institute, Translational Therapeutics (United States), University of Pennsylvania, National Institute of Allergy and Infectious Diseases

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Variable analysis

independent variables

Blimp-1-YFP^lo Tim3^lo (stem-like)
Blimp-1-YFP^hi Tim3^hi (terminally exhausted)
KLRG1^lo (memory precursor)
KLRG1^hi (short-lived effector)

dependent variables

Chromatin immunoprecipitation (ChIP) analysis

control variables

P14 cells
More than ten mice from each infection were pooled for each sort
Two biological replicates of each CD8+ T cell population were collected
DNA-protein crosslinking, nuclei isolation, and chromatin sonication were performed using truChIP Chromatin Shearing Kit (Covaris) according to manufacturer's instructions
Immunoprecipitation by anti-H3K27Ac (ab4729, AbCam)
ChIPmentation was performed according to the published protocol^50
The libraries were sequenced for 50 cycles (single read) on a HiSeq 3000 (Illumina)

controls

Positive control: Not specified
Negative control: Not specified

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