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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 22, 2022 |
| Title |
Naïve3_mouse_methylation |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: P14 CD8 T cell
sequencing batch: 3
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell sorting was performed by FACS Aria II (BD Biosciences). For RNA-seq of P14s in LCMV Arm or in TRAMPC1-GP bearing mice, Live, CD4-CD14-CD19-, CD45.1+CD8+CD44+PD1+ were sorted. For RNA-seq of endogenous CD8 T cells from tumor, Live, CD4-CD14-CD19-Ly6G-, CD8+CD44+PD1+ 127+ or TIM3+ were sorted. performed by FACS Aria II (BD Biosciences). For RNA-seq of P14s in LCMV Arm or in TRAMPC1-GP bearing mice, Live, CD4-CD14-CD19-, CD45.1+CD8+CD44+PD1+ were sorted. For RNA-seq of endogenous CD8 T cells from tumor, Live, CD4-CD14-CD19-Ly6G-, CD8+CD44+PD1+ 127+ or TIM3+ were sorted. Qiagen micro RNA/DNA all prep columns were used for RNA extraction.
DNA was isolated using the AllPrep DNA/RNA Micro kit (Qiagen) and sonicated to generate random 300 to 500-bp fragments. DNA was end-repaired and A-tailed using the Hyper Prep Kit (KAPA Biosystems) following the manufacturer’s protocol. Sequencing adapters that contained fully methylated cytosine residues (Integrated DNA Technologies) were ligated using the Hyper Prep Kit (KAPA Biosystems). Adapter-ligated DNA was bisulfite converted using the EpiTect Bisulfite Kit (Qiagen), with a denaturation time of 10 min. Final libraries were PCR amplified 8–11 times using Tru-seq (Illunima)-compatible custom index primers, as previously described in (Barwick et al., 2016), and HiFi Uracil (KAPA Biosystems). The resulting WGBS libraries were quality checked by Bioanalyzer, pooled at equimolar ratios and sequenced on a NovaSeq S4
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
P14 TCR transgenic CD8 T cells specific for GP33 epitope of LCMV. Activated P14s sorted from LCMV Armstrong infection, TDLN and tumor of TRAMPC1-GP bearing mice, or naïve spleen.
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| Data processing |
Sequenced data was aligned to the mouse genome using Hisat2 and analyzed using custom R and Python scripts. Fisher’s exact test was used to define regions of statistically (p value less that 1x10-4) differentially methylated CpG motifs. Continuous regions of differentially methylated CpGs were identified by finding regions where at least 6 out of 10 CpGs in a continuous stretch were differentially methylated. These regions were then collapsed and analyzed as single ‘differentially methylated regions’ (DMRs).
Assembly: mm10
Supplementary files format and content: CSV file with raw counts for all genes and samples
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| Submission date |
Dec 20, 2022 |
| Last update date |
Jan 04, 2023 |
| Contact name |
Haydn Kissick |
| E-mail(s) |
haydn.kissick@emory.edu
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| Phone |
6172598364
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| Organization name |
Emory University
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| Street address |
1462 Clifton Rd
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| City |
Atlanta |
| State/province |
Georgia |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE216731 |
LN-stem, tumor stem, tumor terminally differentiated CD8 T cells from human kidney cancer |
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| Relations |
| BioSample |
SAMN32330204 |
| SRA |
SRX18916817 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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