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| Status |
Public on Feb 23, 2022 |
| Title |
iMAC_single cell RNA seq |
| Sample type |
SRA |
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| Source name |
stem cell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: ESCs
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| Growth protocol |
The human ESCs were differentiated into macrophages following a previous protocol with some modifications (PLoS One. 2011;6(7):e22261. doi: 10.1371/journal.pone.0022261). Briefly, ESCs were plated onto matrigel-coated plates. When colonies grew up to 500μm in diameter, cell culture media was replaced with mesoderm differentiation medium (APEL 2 supplemented with 1X Insulin-Transferrin-Selenium-X (ITX, Invitrogen), 100ng/ml BMP4). After 48hrs, 100ng/ml BMP4 was replaced by 20ng/ml BMP4. On day 4, BMP4 was replaced by 40ng/ml VEGF and 50ng/ml SCF. After 2 days, medium was changed by hematopoietic differentiation medium (APEL 2 supplemented with 1X ITX, 50ng/ml SCF, 10ng/ml TPO, 50ng/ml IL-3, 50ng/ml IL-6 and 50ng/ml Flt-3L). On day 15, floating cells were collected, and incubated in macrophage differentiation medium (RPMI 1640 supplemented with 100ng/ml M-SCF).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cell capturing and barcoding to generate single-cell Gel Beads-in Emulsion (GEMs) was performed using the 10x Genomics Chrominum platform following the manufacturer’s protocol. In Brief, cell suspensions were loaded onto 10x Genomics Single Cell 3’ Chips with the reverse transcription master mix with RT Primer (TSO) (PN-310354). In this step, cells were separated into the GEMs along with gel beads coated with oligonucleotides which allow the mRNA capture inside the droplets by 30 bp oligo-dT after cell lysis and, provide barcodes to index cells (16 bp) with transcripts (10 bp UMI). After reverse transcription (RT), the barcoded cDNAs were amplified.
library was generated using the Single Cell 3’ Reagent Kit (v2 chemistry). We used illumine Hiseq 4000 system in stand-alone mode to sequence the libraries to obtain pair end sequencing 26 bp (read1) X 98 bp (read2). To process the barcodes, UMI counting and demultiplexing, we used the official 10x Genomics pipeline Cell Ranger v2.1.1. The raw base call files generated by Illumina sequencers were demultiplexed into reads in FASTQ format using the bcl2fastq v.2.20 (GEO number, Data Citation 1). The raw reads were then trimmed from the 3’ end and, get the recommended number of cycles for read pairs (Read1: 26 bp; Read2: 98 bp). The reads of each library were processed separately using the “cellranger count” pipeline to generate a gene-barcode matrix for each library. In this step, the reads were aligned to a human reference genome (version: hg19, GRCh37.p13).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
The reads of each library were processed separately using the “cellranger count” pipeline to generate a gene-barcode matrix for each library. In this step, the reads were aligned to a human reference genome (version: hg19, GRCh37.p13).
The concatenated gene matrix, barcode matrix and count matrix were imported into Seurat (http://satijalab.org/seurat/) to undergo data preprocessing.
We filtered genes whose expression was detected in fewer than 3 cells. We filtered cells whose expression was detected in less than 100 genes and less than 10 percent mitochondrial genes.
We normalized the read counts for each cell are divided by the total counts for that cell and multiplied by the scale factor. After normalization, the read count data was log- transformed (log (read count + 1)), data scaling using linear models, focused on each minus the mean expression of the gene in all cells.
Genome_build: hg19, GRCh37.p13
Supplementary_files_format_and_content: tab-delimited text files include read count values for each Sample
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| Submission date |
Jul 08, 2019 |
| Last update date |
Feb 23, 2022 |
| Contact name |
Sang Cheol Kim |
| E-mail(s) |
kimsc77@gmail.com
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| Phone |
+82-43-719-8861
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| Organization name |
Korea National Institute of Health
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| Lab |
Division of Bio-Medical Informatics
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| Street address |
Osong Health Technology Administration Complex, 187, Osongsaengmyeong 2-ro, Osong-eup
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| City |
Cheongju-si |
| ZIP/Postal code |
28159 |
| Country |
South Korea |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE133935 |
Single-Cell RNA-sequencing of human embryonic stem cell-derived macrophages |
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| Relations |
| BioSample |
SAMN12230310 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3930366_barcodes.tsv.gz |
13.1 Kb |
(ftp)(http) |
TSV |
| GSM3930366_genes.tsv.gz |
251.2 Kb |
(ftp)(http) |
TSV |
| GSM3930366_matrix.mtx.gz |
13.2 Mb |
(ftp)(http) |
MTX |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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