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| Status |
Public on Nov 29, 2018 |
| Title |
scTrioSeq2Rna_CRC01_LN1_174 |
| Sample type |
SRA |
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| Source name |
CRC
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| Organism |
Homo sapiens |
| Characteristics |
disease state: Carcinoma
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| Treatment protocol |
Informed consent was obtained from all patients preoperatively. Multi-regional sampling tissues were collected. For each tumor, 3-6 regions, including both surface and center areas, were sampled. Except for the MP of CRC01 which was sampled after six cycles of chemotherapy, other tumors were all sampled before treatment. Patient-derived tumors and adjacent normal tissue were collected and processed immediately after surgical resection. The dissected tissues were then mechanically dissociated and enzymatically digested to single-cell suspension using collagenase type II (Invitrogen, cat. #17101015) and collagenase type IV (Invitrogen, cat. #17104019). For 5 patients (CRC11, CRC12, CRC13, CRC14, and CRC15), leukocytes were depleted by magnetic-activated cell sorting (MACS) (CD45 Microbeads, Miltenyi Biotec, cat. #130-045-801) or fluorescence-activated cell sorting (FACS) (BV421 Mouse Anti-Human CD45, BD Horizon, cat. #563879).
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| Growth protocol |
The HEp-2 (ATCC CCL-23) cell line were cultured in RPMI 1640 with 10% FBS, and digested with trypsin before single cell picking.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The single viable cells were individually picked into 200-uL tubes containing lysis buffer. For scTrio-seq2, we used magnetic beads (Invitrogen, cat. #65011) to separate the nucleus and RNA of one single cell. We added 0.2 ?L magnetic beads to each single-cell lysis buffer. Then the single cells were lysed and vortexed for 1 min to release RNA. The lysis products were then centrifuged at 1,000 × g for 5 min at 4°C, and placed on the magnetic rack for 5 min. The magnetic beads can aggregate on the surface of the nucleus to maintain the nucleus in the pellet, while the RNA was released in the supernatants. The supernatants containing RNA were transferred to a new tube for transcriptome sequencing. The remaining beads containing a single nucleus were re-suspended with lysis buffer of scBS-seq for DNA methylation sequencing
For single-cell whole-genome bisulfite sequencing, the scBS-seq libraries were constructed according to the published protocol as described in 2017 (Clark et al., 2017). For CRC01 and CRC02, the transcriptome sequencing libraries were constructed according to Tang protocol (Tang et al., 2009); for the remaining patients, the transcriptome sequencing libraries were constructed according to a multiplexed scRNA-seq method, in which the poly T primers were combined with barcodes and unique molecule identifiers (UMIs) (Dong et al., 2018).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
single cell RNA seq
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| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
The raw sequencing reads were trimmed to remove sequencing adapters, amplification primers, and low-quality bases in read ends.
For the data from scRNA-seq performed according to Tang protocol (Tang et al., 2009), trimmed clean reads were aligned to human reference genome (hg19) using STAR (v2.5.0) 2-pass mapping steps. For the data from scRNA-seq performed according to the multiplexed scRNA-seq method, the analyses were done as the previous paper (Dong et al., 2018).
For bisulfite sequencing data, trimmed clean reads were mapped to human reference genome (hg19) using Bismark (v0.7.6)
Genome_build: hg19
Supplementary_files_format_and_content: The FPKM.txt files include the FPKM values of RefSeq genes. The TPM.txt files include the TPM values of RefSeq genes. The SingleC files include the DNA methylation level of each singleC sites.
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| Submission date |
Jul 08, 2017 |
| Last update date |
Nov 30, 2018 |
| Contact name |
Yu Hou |
| Organization name |
Peking University
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| Department |
School of life sciences
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| Lab |
Fuchou Tang
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| Street address |
No.5 Yiheyuan Street
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE97693 |
Single-cell Multi-omics Sequencing and Analyses of Human Colorectal Cancer |
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| Relations |
| BioSample |
SAMN07335234 |
| SRA |
SRX3002205 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2696965_scTrioSeq2Rna_CRC01_LN1_174.FPKM.txt.gz |
95.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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