title: GSM4293893: 383227: Healthy Donor TCM WGBS; Homo sapiens; Bisulfite-Seq
library_construction_protocol: DNA was extracted followed by bisulfite treatment for library prepration. Nuclei were exteacted for ATAC-Seq. Libraries were prepared according to Illumina's instructions accompanying the TruSeq DNA Methylation Kit (Part# EGMK81312). Briefly, Bisulfite converted DNA was annealed with random hexamer with tagging sequence. DNA copy was synthesized using TruSeq DNA Methyl Polymerase. Then Exonuclease I was used to digest excess random primer.  The 3' ends of the strands are then selectively tagged with a second specific sequence tag to generate di-tagged DNA using DNA polymerase. The di-tagged DNA with known sequence tags at both ends was cleaned up by the AMPureXP Beads. The dsDNA library was generated by PCR amplification with Illumina primers for 10 cycles and library fragments of ~350 bp (insert plus adaptor and PCR primer sequences). The library was purified by the AMPureXP Beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2500 following the manufacturer's protocols. For ATAC-Seq, nuclei were isolated from FACS purified tetramer+ beta cell-specific CD8 T cells and polyclonal Tem and naive CD8 T cells from T1D donors using the Low Cell Input Nuclei Isolation protocol from 10x Genomics. The transposition reaction was then performed on the bulk nuclei. The transposed nuclei were partitioned into nanoliter-scale barcoded Gel Beads-in-emulsion (GEMs) after running the loaded Chromium microfluidic Chip E on the Chromium Controller, and the transposed DNA was uniquely indexed and barcoded for each individual nucleus per manufacturer's instructions (10x Genomics). Libraries were generated and sequenced using Illumina Hiseq system following the manufacturer's protocols. For each sample, the single cell ATAC data were mapped to hg38 using the Cell Ranger ATAC pipeline.  We then used SnapATAC (https://github.com/r3fang/SnapATAC) to combine multiple samples, i.e. the fragment.tsv files generated by Cell Ranger ATAC pipeline, and performed the clustering analysis.
attributes.tag: GEO Accession
attributes.value: GSM4293893