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| Status |
Public on Jul 01, 2021 |
| Title |
sc-mCherry_2 L2 |
| Sample type |
SRA |
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| Source name |
embryonic
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| Organism |
Homo sapiens |
| Characteristics |
gfp: GFP-
mcherry: mCherry+
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| Growth protocol |
Differentiation was initiated 72 hours after plating when the culture was approximately 80% confluent. Step 1: Cells were differentiated with 1.5 μM CHIR99021, 20 ng/mL BMP4 and 20 ng/mL Activin A in RPMI supplemented with B27 minus insulin, 2 mM GlutaMAX, 1x NEAA and 1x Pen/Strep for 3 days . Step 2: For cardiomyocyte differentiation, cells were treated for an additional 3 days with 5 µM XAV939 . For hPSC-SAN differentiation, Step 1 was followed by addition of 0.1 μM cucurbitacin, 1 μM retinoic acid, 5 μM SU5402 in RB27-INS from day 3-6. 5 µM XAV939 was added from day 5-6. From day 6 onward, both cardiomyocyte and hPSC-SAN differentiation were carried out in RPMI supplemented with B27, 2 mM GlutaMAX, 1x NEAA and 1x Pen/Strep (RB27+INS). Step 3: hPSC-SAN differentiation included additional treatment with 5 µM Tyrphostin AG 490 (Sigma Aldrich) from day 6-9 in RB27+INS. The HDAC inhibitor, chidamide, was added to the differentiation cocktail from day 7-9 with a final concentration 5 µM.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
scRNA-seq libraries were prepared following the Dropseq protcol (Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, and McCarroll SA. Highly Parallel Genome- wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell, 161(5):1202–1214, 2015. doi: 10.1016/j.cell.2015.05.002.)
Nextera XT Library Prep Kit
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Dropseq
mCherry-pool2.dge.txt.gz
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| Data processing |
Illumina bcl2fastq2 v2.19 was used for basecalling.
All single-cell RNA-seq samples were processed according to the Drop-seq Computational Protocol with the Drop-seq Toolkit v1.2 created by the McCarroll Lab (http://mccarrolllab.com/dropseq/). Sequences were first tagged with their corresponding cell and molecular barcodes (unique molecular identifiers, UMI) using dropseq TagBamWithReadSequenceExtended. Cell and molecular barcodes were required to have a minimum base quality of 10 across their lengths, otherwise they were discarded. SMART adapter sequences were trimmed from the 5’ end of each read whenever there were at least five continuous bases exactly matching the SMART adapter (dropseq TrimStartingSequence). Putative poly(A) tails were trimmed from the 3’ end whenever there were stretches of at least 6 adenosines with zero mismatches (dropseq PolyATrimmer).
Barcode-tagged and cleaned reads were aligned with default parameters of STAR (v.2.4.2a) after conversion of the unaligned BAM file to FASTQ format (Picard's SamToFastq tool). The final file containing alignment information plus the cell and UMI tags were generated using Picard MergeBamAlignment merging the unaligned and aligned BAM files to restore the XC and XM barcodes. Sequences that mapped to more than one locus were excluded from further analysis by filtering reads with a mapping quality lower than 10.
To determine the numbers of detected transcripts per gene and cell, the overlaps of the aligned reads with gene annotation (Ensembl release 76) were counted and summarized using dropseq TagReadWithGeneExon and dropseq DigitalExpression. Individual gene expression matrices from each sample were combined in R to create a single gene count matrix for all cells and genes.
Genome_build: GRCh38
Supplementary_files_format_and_content: excel file including gene name, Ensembl gene id, RPKM value for each each gene in each sample
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| Submission date |
Sep 07, 2018 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Shuibing Chen |
| E-mail(s) |
shuibing.chen@gmail.com
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| Phone |
2127465431
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| Organization name |
Weill Cornell Medical College
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| Department |
Surgery
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| Street address |
A 827B, 1300 York Ave
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE118087 |
Pharmacogenetics and Drug Discovery for Anthracycline-Induced Cardiotoxicity Enabled by Sinoatrial Node-like Cells Derived from Human Pluripotent Stem Cells |
| GSE119682 |
Pharmacogenetics and Drug Discovery for Anthracycline-Induced Cardiotoxicity Enabled by Sinoatrial Node-like Cells Derived from Human Pluripotent Stem Cells [scRNA-Seq] |
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| Relations |
| BioSample |
SAMN09992596 |
| SRA |
SRX4660097 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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