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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.
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|
|
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.
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|
|
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 |