|
| Status |
Public on Jul 30, 2022 |
| Title |
V19-4 |
| Sample type |
SRA |
| |
|
| Source name |
patient 19 adjacent normal tissue region 1
|
| Organism |
Homo sapiens |
| Characteristics |
subject id: patient 19
patient diagnosis: non-small cell lung cancer (NSCLC)
tissue: adjacent normal tissue
|
| Treatment protocol |
Fresh lung tumor tissues and adjacent normal tissues were dissected in RPMI-1640 medium + 10% FBS on ice, and washed with ice-cold DPBS. Then transfer the tissues to a tube , cut it into small pieces with a scissor and resuspend it in 1ml of digestion buffer consisting of Collagenase Type I(2mg/ml; Gibco), Dispase II(1mg/ml; Millipore) and DNase I (0.2mg/ml; Roche) in RPMI-1640 medium. After incubation at 37°C for 30-40 min with frequent agitation, 500ul of RPMI-1640 medium was added to stop digestion. The tissues were pipetted up and down for 40-50 times and the cell suspension was filtered through a 100-um mesh filters. Then the cells were centrifuged at 500g for 10 min at 4°C, the pelleted cells were re-suspended in red blood cell lysis buffer and incubated at RT for 3min. The cell pellets were washed again and re-suspended with RPMI-1640 medium +10 % FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Briefly, single cells were picked into the RNA lysis buffer by a mouth pipette. Then disrupt the cells by vigorously vortexing for 40 seconds, and the released RNAs were incubated at 72℃ for 3min to open the secondary structure. The remaining cell suspension was centrifuged at 500g for 10min and the supernatant was discarded. Then the cell pellets were used for genomic DNA extraction with DNeasy Blood & Tissue Kit.
Single-cell cDNA amplification was based on a modified STRT-seq protocol. Briefly, the released mRNAs were reverse transcribed into cDNA by poly (T) primers anchored with 8bp cell-specific barcodes and 8bp unique molecular identifiers (UMIs), followed by second strand cDNA synthesis and pre-amplification. The amplified cDNA from different cells can be pooled together, fragmented to around 300bp, and the 3’ ends were enriched by the affinity interaction with Dynabeads® MyOne™ Streptavidin C1 beads for library construction using Kapa Hyper Prep Kit. The constructed libraries were sequenced on Illumina HiSeq 4000 system as paired-end 150bp reads.
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| 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 were used for sequence alignment and uniquely mapped reads were kept.
Genome_build: Reference sequence and transcript annotation files were from UCSC genome assembly hg19
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each single cell RNA samples
Supplementary_files_format_and_content: The *Info.txt files at the foot of each sample page include the cell barcode sequence and its associated ID.
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| |
|
| Submission date |
Sep 13, 2018 |
| Last update date |
Nov 08, 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) |
| GSE119911 |
Comprehensive transcriptomic profiles of non-small cell lung cancer by single-cell RNA-seq |
|
| Relations |
| BioSample |
SAMN10055778 |
