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| Status |
Public on Jan 20, 2015 |
| Title |
5076_CD4_Memory_48 [Bisulfite-Seq] |
| Sample type |
SRA |
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| Source name |
Primary Memory CD4 T Cell
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| Organism |
Homo sapiens |
| Characteristics |
activation: anti-CD3/CD28 beads, 48 hours
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| Treatment protocol |
Purified naïve and memory CD4 T cells were cultured in RPMI supplemented with 10% FBS, 100 U/ml penicillin and 100mg/ml streptomycin at 37C and 5% CO2 with anti-CD3/CD28 activator beads (Invitrogen) for 0 or 48 hours.
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| Growth protocol |
Naïve and memooy CD4 cells were negatively selected using magnetic beads (Stem Cell Technologies). CD8 T cells and CD19 B cells were positively selected on magnetic beads (Miltenyi Biotec).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was prepared from cell pellets using an All Prep kit (QIAGEN). DNA was bisulfite converted using the EpiTect Bisulfite conversion kit (QIAGEN).
Bisulfite treated genomic DNA was amplified by microdroplet PCR amplification on an RDT 1000 (RainDance Technologies) with a custom primer library according to the manufacturer’s instructions. Briefly, microdroplets containing genomic DNA were merged with microdroplets from the custom primer droplet library. The custom primer droplet library consists of a collection of individual primer droplets, where each primer droplet contains matched pairs of forward and reverse primer (for each amplicon defined within the primer library. For primers covering a CpG, a degenerate base (C/T or G/A, depending on the strand) was used at that location. The RDT1000 generated each PCR droplet by pairing a single genomic DNA template droplet with a single primer droplet. Paired droplets flow past an electrode embedded in the chip and are instantly merged together. All resulting PCR droplets were automatically dispensed as an emulsion into a single PCR tube and transferred to a standard thermal cycler for PCR amplification. Each sample generated more than 1 million singleplex PCR droplets. Samples were amplified for 55 cycles, the droplet emulsion was broken, and the PCR product was purified over a MinElute column (QIAGEN) following the manufacturer’s recommended protocol. RainDance PCR products were end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. End-repaired products were concatenated using T4 DNA ligase and then fragmented to 200 bp using a Covaris S2. Fragmented PCR product was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified for 15 cycles and library fragments of ~350 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on an Illumina HiSeq 20002000 following the manufacturer's protocols. 100 bp single-end reads were generated with 10 samples per lane.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Bisulfite Treated
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| Data processing |
Basecalls performed using CASAVA versions 1.5-1.8.1
Bisulfite reads were mapped to hg18 with novoalign in bisulfite mode with the following parameters: novoalign -d [index file] -f [fastq file] -F STDFQ -b 4 -c 8 -a -h 120 -t 240 -s 50 -o SAM
Genome_build: hg18
Supplementary_files_format_and_content: Tab delimited text files containing % methylation by CpG with counts for each base at the indicated position. Headers are "Gene, Chr, Position, Total_FWD_Reads, FWD_A, FWD_C, FWD_G, FWD_T, %Methylation_FWD, Total_REV_Reads, REV_A, REV_C, REV_G, REV_T, %Methylation_Rev"
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| Submission date |
Jul 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel Salomon |
| E-mail(s) |
[email protected]
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| Organization name |
The Scripps Research Institute
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| Department |
Molecular & Experimental Medicine
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| Lab |
Salomon
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| Street address |
10550 N Torrey Pines Rd, MEM-241
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE59858 |
Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation [Bisulfite-Seq] |
| GSE59860 |
Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation |
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| Relations |
| BioSample |
SAMN02943947 |
| SRA |
SRX665080 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1448223_mapped_Run_H19_BC4_TGACCA_L001_R1_001.fastq.gz-methylation-at-each-cpg-no_primer.txt.gz |
650.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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