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| Status |
Public on Feb 10, 2018 |
| Title |
GW19_PFC1_2 |
| Sample type |
SRA |
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| Source name |
Cells From Prefrontal Cortex
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Prefrontal Cortex
developmental stage: 19 weeks after gestation
gender: female
barcode_id: 1-22,24-33,35-42
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| Treatment protocol |
To maintain the cell activity, we dissected all prefrontal cortex from nine fetal brain in ice cold artificial cerebrospinal fluid containing 125.0 mM NaCl, 26.0 mM NaHCO3, 2.5 mM KCl, 2.0mM CaCl2, 1.0 mM MgCl2, 1.25 mM NaH2PO4 at a pH of 7.4 when oxygenated (95% O2 and 5% CO2).The PFC tissue was first digested in brain tissue digestion medium 1 (BTDM1, 2mg/ml collagenase IV (Gibco, Cat.17104-019) and 10 U/μl DNase I (NEB, Cat.M0303L) in hybradate E medium). After brief pipet to roughly digest the tissue into small pieces, brain tissue digestion medium 2 (BTDM2, 1mg/ml papain (Sigma, Cat.P4762) and 10 U/μl DNase I in hybradate E medium).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cells were directly picked into 2.5ul cell lysis buffer the same as Smartseq2 except that we used barcoded reverse transcription primers instead of oligdT30VN primer. Further cell lysis, reverse transcription and amplification was referred to Smartseq2 with little modification.
Each cell was amplified for 18 cycles in total. The PCR product for each barcode 1 to barcode 96 was pooled together for purification and library construction. The libraries were sequenced on illumina platform for 1.8M 150bp pair-end reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
polyA RNA
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| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
The first 8bp of read2 were used to split the mixed data into single cells according to the barcode sequences. The following 8bp were recorded and attached to the end of each read2 as unique molecular identifier (UMI). After that, reads1 were separated by the header of the corresponding read2 and then added UMI information. Hereafter, the split reads1 were trimmed off ploy(A) tail sequence and template switch oligo (TSO) sequence. Then we did quality control by removing out reads with adaptor contaminants (length < 37bp) and low-quality bases (N > 10%).
Subsequently, TopHat (version 2.0.12) was applied to align clean reads to the hg19 human transcriptome downloaded from UCSC. Uniquely aligned reads were counted by the tool ‘htseq-count’ of HTSeq. According to the UMI information, transcripts with same UMI sequence of each gene were merged as one, thus the number of different UMIs for each gene was considered as the transcript counts of that gene without PCR bias for each sequenced cell.
The transcript counts of each cell were normalized to transcript per million (TPM), and the formula to calculate TPM is: the transcript counts of each gene divided by the sum of transcript counts of that cell, and multiplied by one million. TPM values were then normalized by log2(TPM/10 + 1) for the following analysis, since the sequencing depth of most single cell samples were less than 1,000,000 reads, we divided TPM values by 10.
Genome_build: hg19
Supplementary_files_format_and_content: xls files which include the TPM values of RefSeq genes.
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| Submission date |
Dec 11, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Shu Zhang |
| E-mail(s) |
zhshu0917@pku.edu.cn
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| Organization name |
BIOPIC
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| Lab |
Fuchou Tang
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| Street address |
Peking University, No.5 Yiheyuan Road Haidian District
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE104276 |
Single-cell RNA-Seq reveals a developmental atlas of human prefrontal cortex |
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| Relations |
| BioSample |
SAMN08162147 |
| SRA |
SRX3462657 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2884078_GW19_PFC2.UMI_TPM_no_ERCC.txt.gz |
534.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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