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| Status |
Public on May 04, 2022 |
| Title |
MULTIseq Sample A |
| Sample type |
SRA |
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| Source name |
MULTIseq pooled samples
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| Organism |
Homo sapiens |
| Characteristics |
sample type: mixed pool
cell type: hiPSC derived cells
cell type: FACs-sorted
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| Treatment protocol |
Monolayer cardiac differentiation was carried out following a published protocol(Lian et al., 2015). The cardiac organoid differentiation procedure was adapted from a protocol described Previously (Branco et al., 2019).
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| Growth protocol |
Two WTC lines (ACTN2-eGFP,Myl2-eGFP) and SCVI114 line were maintained in completely defined albumin-free E8 medium (DMEM/F12 with L-glutamine and HEPES, 64μg/ml L-Ascorbic Acid-2-phosphate, 20μg/ml insulin, 5μg/ml transferrin, 14ng/ml sodium selenite, 100ng/ml FGF2, 2ng/ml TGFb1) on Matrigel coated tissue culture plates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cardiac cells from monolayer differentiation culture at day 15 and day 30 were dissociated with TrypLE and washed once with HBSS-/- (Ca2+/Mg2+ free). The cardiac organoids were sequencially incubated with 0.25% Trypsin/EDTA and a collagenase HBSS+/+ mixture before filtered through a 40 μm filter. The cells from each sample were stained with with a unique MULTI-seq barcode separately and pooled before library preparation.
The pooled cells were captured in 10X Chromium (10X Genomics, 120223) by following the single cell 3’ reagent kits v3 user guide. Briefly, cells were loaded into each chip well to be partitioned into gel beads in emulsion (GEMs) in the Chromium controller. We targeted for 25,000 cells in each chip well and profiled one well for the first batch experiment and two chip wells for the second experiment. The cells were then lysed and barcoded reverse transcribed in the GEMs. After breaking the GEMs and further cleanup and amplification, the cDNA was enzymatically fragmented and 3′ end fragments were selected for library preparation. After further processing including end repair, A-tailing, adaptor ligation, and PCR amplification, a string of sequences including sample index, UMI sequences, barcode sequences, and sequencing primer P5 and P7 were added to cDNA on both ends. The libraries were sequenced on Illumina HiSeq X platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Reads were processed using the Cell Ranger (version 3.1.0) count pipeline using the human reference genome GRCh38.p12, annotations from Ensembl (version 93) with the arguments --chemistry= SC3Pv3, --expect-cells=10,000 or 25,000, depending on the specific library. For MULTIseq fastq files, library_type was set to "Custom".
Sample demultiplexing was performed using the R package deMULTIplex (version 1.0.2) [https://github.com/chris-mcginnis-ucsf/MULTI-seq].
Quality control and filtering were performed using the R package scater [10.1093/bioinformatics/btw777] (v1.14.6). For each sample, cells with total library size >=2000, number of detected genes >=1000 and <= 8000, and <=30% percentage of mitochondrial reads were considered. Additionally doublets with the same MULTI-seq barcode were filtered using the scds R package [10.1093/bioinformatics/btz698] (v1.2.0) as described in Supplementary Note XYZ.1. Only genes with at least 1 count in at least five cells were considered and log-normalized counts were calculated using the deconvolution method of the scran R package [10.12688/f1000research.9501.2].
The sample metadata, barcodes, counts matrices, and log-normalized counts are additionally provided as processed data files.
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: H5, mtx, txt and csv files.
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| Submission date |
Dec 21, 2020 |
| Last update date |
May 04, 2022 |
| Contact name |
Dennis Kostka |
| E-mail(s) |
kostka@pitt.edu
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| Organization name |
University of Pittsburgh
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| Department |
Developmental Biology
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| Street address |
530 45TH ST
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15201 |
| Country |
USA |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE163619 |
Multiplexed single cell mRNA sequencing of monolayer- and organoid-system differentiated cells derived from human iPSC with and without NKX2-5 genetic manipulations |
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| Relations |
| BioSample |
SAMN17128295 |
| SRA |
SRX9728574 |
| 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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