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Sample GSM2976284 Query DataSets for GSM2976284
Status Public on Jul 30, 2022
Title P3-PT1
Sample type SRA
 
Source name Patient 3 primary tumour
Organism Homo sapiens
Characteristics patient id: 3
patient disease: colorectal cancer, liver metastasis
tissue: primary tumour
Treatment protocol Normal and cancer colon tissues from six patients were dissected and washed by PBS for several times. Then the tissues were cut into small pieces by surgical scissors. Then the 2mg/mL Collagenase/Dispase was used to digest the tissues into single cells at 37℃ for 30-40 min after which the cell suspension was centrifuged at 800g at 4℃ for 5min. The supernate was discarded and 1% HSA was used to re-suspended the cell pellet.
Extracted molecule total RNA
Extraction protocol Single cells were picked into scRNA-seq lysis buffer by using mouth pipette. Then the cells in scRNA-seq lysis buffer were vortexed for 45 second and heated at 72℃ for 3min to release RNA. Bulk genomic DNA were extracted according to the manufacturer protocol of DNeasy Blood & Tissue Kit (QIAGEN).
Modified STRT-Seq protocol: we modified the STRT-Seq protocol for application to a large number of cells. Briefly, the 8bp barcodes was used to label individual cells. So the cDNA of cells with different barcodes can be pooled together for library constructions. And we also enrich the 3’ cDNA by the affinity interaction between biotin and Dynabeads® MyOne™ Streptavidin C1 beads. Then the fragmented DNA inserts were library constructed according to the manufacturer procedures of KAPA Hyper Prep Kits with PCR Library Amplification/Illumina series (KAPA, KK8054) and sequenced on Illumina 4000. Bulk genomic DNA library construction protocol: The extracted genomic DNA was fragmented by sonication into 300bp and then library constructed according to the manufacturer procedures of KAPA Hyper Pre.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Description PolyA RNA
Single cell RNA-Seq.
processed data file: metastatic_Colon_TPM.txt
Data processing For data from Illumina, Illumina CASAVA version 1.8 were used to the basecalling.
scRNA-Seq: Read 2 was used to obtain the cell barcodes to further split the reads according to the cell IDs (barcode) and the same time recorded the UMI sequences; Read 1 was picked in each cell according to the cell ID in read2 and the UMI information was aligned to it, and then these raw reads were trimmed to remove TSO or polyA sequence. Adaptor contamination and low-quality reads were discarded from the trimmed Read 1 raw data. TopHat (version 2.0.14) with default settings was used for sequence alignment and uniquely mapped reads were kept.
Whole genome sequencing: Quality control pipeline was firstly performed on paired-end sequencing data to remove low quality reads. The cleaned reads were mapped to human genome (hg19) by BWA. The total sequence reads of each 5M window were calculated and normalized by the total read depth of each samples. For each 5M window, they were normalized by dividing the average reads depth of all samples. Finally, the CNV values were visualized by ggplot2 package.
Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly hg19.
Supplementary_files_format_and_content: metastatic_Colon_TPM.txt: Tab-delimited text file includes TPM values for each Sample.
Supplementary_files_format_and_content: 5M_Merge_Reads.txt: Tab-delimited text file includes read depth per 5M window for each bulk tissue.
Supplementary_files_format_and_content: The *.txt files provide the barcode sequences for the samples.
 
Submission date Feb 01, 2018
Last update date Jul 30, 2022
Contact name Rui WANG
E-mail(s) fish_cat_wr@sina.cn
Phone 15801166445
Organization name Peking University
Department Biodynamics Optical Imaging Center (BIOPIC)
Lab Fuchou Tang
Street address No.5 Yiheyuan Road, Haidian District
City Beijing
ZIP/Postal code 100871
Country China
 
Platform ID GPL20301
Series (1)
GSE110009 Single cell transcriptome landscape of primary and metastatic tumor in colon cancer
Relations
BioSample SAMN08452028
SRA SRX3639795

Supplementary file Size Download File type/resource
GSM2976284_P3-PT1.txt.gz 632 b (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record
Processed data provided as supplementary file

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