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Sample GSM4743234

Query DataSets for GSM4743234

Status

Public on Dec 16, 2021

Title

MM.P20181219

Sample type

SRA

Source name

Organism

Homo sapiens

Characteristics

tissue: Tumor
platform: 10X

Extracted molecule

polyA RNA

Extraction protocol

Tumors and adjacent normal tissues were cut into approximately 1-2 mm3 pieces in the RPMI-1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco), and enzymatically digested with gentleMACS (Miltenyi) for 60 min on a rotor at 37°C, according to manufacturer’s instruction. The dissociated cells were subsequently passed through a 100 µm SmartStrainer and centrifuged at 400 g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (TIANDZ) and incubated on ice for 1-2 min to lyse red blood cells. After washing twice with 1× PBS (Gibco), the cell pellets were re-suspended in sorting buffer (PBS supplemented with 1% FBS).
Single cell suspensions were stained with antibodies against CD45 and 7AAD for FACS sorting, performed on a BD Aria SORP instrument. Based on FACS analysis, single cells were sorted into 1.5 ml tubes (Eppendorf) and counted manually under the microscope. The concentration of single cell suspensions was adjusted to 500-1200 cells/ul. Cells were loaded between 7,000 and 15,000 cells/chip position using the 10x Chromium Single cell 5’ Library, Gel Bead & Multiplex Kit and Chip Kit (10x Genomics, V3 barcoding chemistry) according to the manufacturer’s instructions. All the subsequent steps were performed following the standard manufacturer’s protocols. Purified libraries were analyzed by an Illumina Hiseq X Ten sequencer with 150-bp paired-end reads.
10x Chromium Single cell 5’ + VDJ Library, and Smart-seq2 Library (only for one CHOL sample)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

processed data file:
MM_10X.CD4.counts.txt.gz
MM_10X.CD8.counts.txt.gz
metadata.txt.gz
10X_VDJ.merge.txt.gz
bulk_RNA.counts.txt.gz
bulk_Exome.mutation.txt.gz

Data processing

For 10X data, Cell Ranger 3.0.0 was used to quantify gene expression level and to identify TCR sequences.
For Smart-seq2 data, HTSeqGenie pipeline (R package version 4.8) was used to quantify gene expression level.
For bulk RNAseq data, UCSC Xena Toil RNAseq pipeline was used to quantify gene expression level.
For bulk exome data, BWA-MEM algorithm was used to align the reads to genome, PICARD was used to sorted and de-duplicated the bam files, GATK 3.7 was used to realign reads around INDEL region and re-calibrate base quality, and finally strelka 1.0.14 was used to call somatic mutations (SNV and INDEL).
Genome_build: GRCh38 for RNA expression and TCR data, and b37 for DNA mutation calling.
Supplementary_files_format_and_content: text files for expression matrices, cell metadata, TCR summary, and mutation summary.

Submission date

Aug 24, 2020

Last update date

Dec 16, 2021

Contact name

Shishang Qin

E-mail(s)

[email protected]

Organization name

Peking university

Street address

No.5 Yiheyuan Road, Haidian District