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| Status |
Public on Jun 29, 2018 |
| Title |
MT_22WF_B1 |
| Sample type |
SRA |
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| Source name |
Human Embryonic Cortex Cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
developmental stage: 22 week gestation
gender: female
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| Extracted molecule |
total RNA |
| Extraction protocol |
A modified Smart-seq2 protocol was applied for single-cell RNA-seq. Briefly, a single cell was picked into the lysis buffer by mouth pipette. The reverse transcription reaction was performed with 24 oligo (dT) primer anchored with the 8 bp cell specific barcode, and also with 8 bp unique molecular identifiers (UMIs).
After the first-strand reaction, the cDNA was amplified by 17 cycles. Then the amplified cDNA of the cells of the same fetal sample were pooled together to proceed the following steps. Biotinylated pre-indexed primer was used to amplify the PCR product by additional 4 cycles to introduce biotin tags to the 3’ end of the amplified cDNA. About 300 ng DNA was sheared to around 300 bp by Covaris S2 and the 3’ ternimal of the DNA was captured by Dynabeads® MyOne™ Streptavidin C1 beads (Thermo Fisher). RNA seq library was constructed using Kapa Hyper Prep Kit (Kapabiosystems) and subjected to 150 bp paired-end sequencing on illumina HiSeq 4000 platform (sequenced by Novogene).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
polyA RNA
MT_22WF_B1
The brain region of each cell is encoded in the column header of the processed data file. Please see the file "Region_Sample_Barcode_Information.xlsx" for a key to the brain regions abbreviations.
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| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
Raw reads were first organized by specific cell barcode information attached in the read 2 of pair-ended reads. Second, unique molecular identifier (UMI) information were added into the corresponding read 1 which were then trimmed to remove template switch oligo (TSO) sequence and polyA tails. Subsequently, we also removed reads with adapter contaminants and low-quality bases (N>10%).
Third, the cleaned reads were aligned to the hg19 human transcriptome (UCSC) using Tophat (version 2.0.12) (Trapnell et al., 2009). Fourth, uniquely mapped reads were counted using htseq-count of the HTSeq package and grouped by cell-specific barcode (Anders et al., 2014). Based on UMI information, duplicated transcripts of each gene were removed. Finally, within a given cell, distinct UMIs of each gene were counted as the transcript number of that gene.
The expression levels TPM, where TPM (transcript-per-million) was calculated by the number of UMIs of each gene divided by all the number of UMIs of a given cell, then multiplying by 1,000,000.
Genome_build: hg19
Supplementary_files_format_and_content: txt files which include the TPM values of RefSeq genes. The cell barcode information is available in the file "Region_Sample_Barcode_Information.xlsx". The columns of the processed data file names are organized as follows: X(region information)_X(22 or 23W(week) F(female) or M(male))_X(batch information)_scX where 'scX' refers to the cell number and can be associated to the barcode on the third tab of "Region_Sample_Barcode_Information.xlsx". For example, sc1 = primer1, sc2 = primer2, etc.
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| Submission date |
Sep 11, 2017 |
| Last update date |
Oct 10, 2024 |
| Contact name |
Ji Dong |
| E-mail(s) |
[email protected]
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| Organization name |
Peking University
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| Street address |
Yiheyuan 5#
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE103723 |
Single cell RNA-seq analysis of human embryonic cortex |
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| Relations |
| BioSample |
SAMN07630965 |
| SRA |
SRX3177313 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2779742_MT_22WF_B1_gene_expression_TPM.txt.gz |
894.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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