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Sample GSM4215321

Query DataSets for GSM4215321

Status

Public on Dec 12, 2019

Title

RNA-seq: P1031_SP

Sample type

SRA

Source name

CD103+ CD39- (SP) T cells isolated from colorectal cancer patient

Organism

Homo sapiens

Characteristics

subject status/id: colorectal cancer patient P1031
age: 79
Sex: female
tissue/position: Colon sigmoideum
cell type: CD103+ CD39- (SP) T cells

Extracted molecule

polyA RNA

Extraction protocol

Peripheral blood mononuclear cells (PBMCs) were isolated using HISTOPAQUE-1077 (Sigma-Aldrich) solution according to the manufacturer’s instructions. Briefly, 3 mL of fresh peripheral blood was collected prior to surgery in EDTA anticoagulant tubes and subsequently layered onto HISTOPAQUE-1077. After centrifugation, lymphocyte cells remained at the plasma-HISTOPAQUE-1077 interface and were carefully transferred to a new tube and washed twice with 1x PBS (Invitrogen). These lymphocytes were re-suspended with FACS buffer (PBS supplemented with 1% fetal bovine serum (FBS, Invitrogen)). Tumors and adjacent normal tissues were cut into approximately 1-mm3 pieces in the RPMI-1640 medium (Invitrogen), and mechanically dissociated and enzymatically digested with MACS Tumor Dissociation Kit (Miltenyi Biotec) for 30 min on a rotor at 37 °C, according to the manufacturer’s instruction. The dissociated cells were subsequently passed through a 70-µm cell-strainer (BD) and centrifuged at 400g for 10 min. The cell pellets were suspended in red blood cell lysis buffer (Solarbio) and incubated on ice for 2 min to lyse red blood cells. After washing twice with PBS (Invitrogen), the cell pellets were re-suspended in FACS buffer. The following fluorescent-labeled antibodies were used: BV711 anti-CD3 (BC96; 1:100—#300450), APC anti-CD8 (RPA-T8; 1:100—#301048), BV421 anti-CD45RA (HI100; 1:100—#47-0458-42), BV421 anti-CD45RO (HI100; 1:100—#47-0458-42), BV421 anti-CCR7 (HI100; 1:100—#47-0458-42), PE anti-CD39 (eBioA1; 1:100—#17-0399-42), FITC anti-CD103 (B-Ly7 and Ber-ACT8; 1:100—#12-1038-42) (all from eBioscience). Cell surface staining was performed in FACS buffer. Stained cells were acquired on the FACS AriaII (all BD Biosciences) for cell sorting. Data were analyzed with FlowJo software (Treestar). 1000 cells of different subtypes including naïve and TEM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO-CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA-CCR7-, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39-, and 7AAD−CD3+CD8+CD103-CD39-, respectively, and sorted into 0.2 ml tubes(Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μl 10 mM dNTP mix (Invitrogen), 1 μl 10 μM Oligo dT primer, 1.9 μl 1% Triton X-100 (Sigma), and 0.1 μl 40 U μl−1 RNase Inhibitor (Takara).
Transcriptome amplifications were performed according to Smart-Seq2 protocol with modification of reagent amount and PCR cycle numbers. The amplified cDNA products were purified with Agencourt XP DNA beads, and the concentration of each sample was quantified by Qubit HsDNA kits. Libraries were constructed and amplified using the TruePrep DNA Library Prep Kit V2 for Illumina. The libraries were then purified with Agencourt XP DNA beads and anaylsed by fragment analyser for quality assessment. Purified libraries were analysed by an Illumina Hiseq 4000 sequencer with 150-bp pair-end reads. Whole-genome bisulfite-seq was performed according to a previously published protocol (45). Briefly, 1000 cells were sorted into lysis buffer by FACS; DNA was released after proteinase treatment at 50?°C and then subjected to bisulfite conversion. After bead-based purification, DNA was complemented with the biotinylated random primer Bio-P5-N9 (5’-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) and 100 units of Klenow polymerase (3’ to 5’ exo-). This random priming was repeated seven times in total. Second strands were synthesized using another random primer, P7-N9 (5’-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3’), and final libraries were generated after 7 to 9 cycles of PCR amplification with the Illumina universal PCR primer and Illumina indexed primer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

processed data file: CD8Tcell_raw_counts.csv.gz

Data processing

kallisto was used to quantify the abundances of transcripts.
DESeq2 was used to conduct differential gene expression analysis
Bismark was used to align sequencing data from Bisulfite-Seq data to human reference genome.
MethPipe was used to quantify the methylation level.
Genome_build: GRCh38
Supplementary_files_format_and_content: raw counts calculated by kallisto; differentially methylated region (DMR) for each CD8 T cell subset

Submission date

Dec 11, 2019

Last update date

Dec 12, 2019

Contact name

Sijin Cheng

E-mail(s)

chengsj@mail.cbi.pku.edu.cn

Organization name

Peking University

Department

School of Life Science