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| Status |
Public on Nov 15, 2023 |
| Title |
Matrigel 3D cultured rLPCs for 8 d (repr_4) |
| Sample type |
SRA |
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| Source name |
Non limb fibroblasts cultured in matrigel 3D culture for 8 days with 4-factor expression
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| Organism |
Mus musculus |
| Characteristics |
cell type: mesenchymal cells
treatment/condition: Matrigel 3D culture, DMEM 10% FBS, Chir99021 (3uM), Fgf8 (150ng/ml), Retinoic acid (25nM), SB431542 (5uM), Y-27632 (10uM)
extract_protocol: 10X v3 scRNA-seq
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| Treatment protocol |
N/A
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| Growth protocol |
Non-limb fibroblasts cultured in 2D, Lentivirally-expressed non-limb fibroblasts (reprogrammed cells) in 3D hyaluronan or matrigel growth culture condition, directly harvested embryonic primary mesenchyme
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| Extracted molecule |
total RNA |
| Extraction protocol |
For sorting reprogrammed Prx1-GFP or Prx1-tdTomato cells from embryos, a FACS sorter Astrios (Beckman Coulter) or On-chip Sort HSG (On-chip Biotechnologies) was used. After washing with PBS, the cells cultured in the HA-gels or on Matrigel were incubated in TryPLE Express (gibco) for 30 min at 37℃. The cell suspension was pipetted with cut P1000 pipette tips every 10 min, to completely dissociate the cell clusters. The suspension was filtrated by 100 μm Cell strainers (Falcon) and 40 μm Cell strainers (VWR), and cells were pelleted by centrifugation (400 x g for 5 min). The pellets were dissociated by DRAQ5/DAPI in 0.1% BSA/PBS and incubated for 5 min before the sorting. DRAQ5-positive, DAPI-negative cells were sorted for cells on reprogramming at day 2, 4, 8. For HA-gel reprogrammed cells at day 14, additional gating on GFP channel derived GFP-positive and GFP-negative samples. For Matrigel-derived day 14 reprogrammed cells for PZL- as well as PZLL- factors, only GFP-positive cells were collected. DRAQ5-positive, DAPI-negative, Matrigel-derived day 8 cultured E9.5 and E10.5 limb progenitors were collected. The E9.5 cultured limb progenitors were subject to 4-OHT, such that large fraction were tdtomato-positive, but the cells were collected regardless of tdTomato-positivity. DRAQ5-positive, DAPI-negative, tdTomato-positive cells were sorted for the limb mesenchyme cells for E10.5, E11.5 as well as E12.5 cells. For E9.5 limb progenitors, samples were collected without tdTomato gating to maximize yield. InDrop cell capture was processed in Harvard Medical School Single Cell Core. All libraries included about 10-15% of Non-limb fibroblasts to mitigate batch effect. Cells were captured by the Chromium Controller (10X genomics, Biopolymer facility at Harvard Medical School).
single cell RNA libraries were prepared for sequencing using 10X Genomics Chromium V3 protocols and inDrops libraries were prepared by the Harvard Single Cell Core according to the published protocol
scRNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
mouse.rds
combined.rds
Matrigel 3D cultured rLPCs for 8 days
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| Data processing |
All four human samples were multiplexed with mouse and chick samples. The chick data was not presented in this manuscript. Thus, for species demultiplexing, Ensembl release 99 hg38 transcriptome, Ensembl release 98 Gallus gallus-6.0 transcriptome and the filtered Ensembl release 98 mm10 transcriptome was merged using the cellranger mkgtf command to generate human-mouse-chick transcriptome for initial mapping for demultiplexing.
For InDrop preprocessing, sequencing results were demultiplexed by dropTag from dropEst package (Petukhov et al. 2018) and demultiplexed reads were aligned with STAR aligner (Dobin et al. 2013). The aligned reads were split into forward and reverse alignment, since InDrop is directional. The resulting forward and reverse alignment files were quantified using dropEst package including directional UMI correction option (Petukhov et al. 2018) with transcriptome annotation split into forward and reverse direction to avoid mapping of antisense reads.
Sequencing results were demultiplexed by cellranger and aligned using cellranger count (internally by STAR aligner (Dobin et al. 2013)). For the four libraries that needed species demultiplexing, cellular barcodes that had less than 5% of UMI counts from other species were selected for subsequent mapping with the corresponding species transcriptome.
Cellular barcodes with high mitochondrial content (>15%), high hemoglobin gene count (>10%) and low gene counts (<1,200) were filered out. All libraries were subject to doublet detection via Scrublet (Wolock et al. 2019). Batch effect was assessed by simply merging the individual UMI count matrices for clustering and Seurat v3 integration procedure was applied (Stuart, Butler et al. 2019) with 30 dimensions for the individual batches. Further, cell cycle effect, fraction of mitochondrial genes were regressed out. Principal component analysis (PCA) was performed on the integrated, scaled features for dimensional reduction and Uniform Manifold Appxoimation and Projection (UMAP) (McInnes et al. 2018) was used primarily for the cellular embedding coordinates. Leiden algorithm was applied on the shared neighborhood graph with 10 iterations (Seurat default) to derive cluster boundaries (Traag et al. 2019).
Genome_build: For all mouse samples, Ensembl release 98 mm10 transcriptome was used as base transcriptome annotation, with pseudogenes filtered from the GTF file using cellranger mkgtf command . For retroviral infected hyaluronan samples, transgenes for human Lin28a, EGFP (for Prx1GFP transgene) was added to generate custom transcriptome annotation for quantification for reprogrammed cells (mm10.98.hlin28a.gtf.gz). For lentiviral infected Matrigel samples, transgenes for EGFP (for Prx1GFP transgene), rtTA, and human Lin41 as well as PLZF, and 3’UTR sequences of WPRE were added to generate custom transcriptome annotation for quantification for reprogrammed cells (mm10.98.4factor.tdtomato.gtf.gz). The limb progenitor cells were subject to the same transcriptome annotation (yielding zero counts for the transgenes). For human-specific mapping, the filtered hg38 transcriptome with transgenes for EGFP, rtTA, and mouse Prdm16 and mouse Lin28a were added (hg38.4factor.gtf.gz).
Supplementary_files_format_and_content: Seurat (v3.1.5) object with meta information; equivalent text file in market matrix format (MTX) with rownames (features) and colnames (barcodes) as well as meta information in tab-delimited format are included. The whole data is split into mouse-only data (prefix mouse.), human-only data (prefix human.), and for convenience, combined data (prefix combined.) which only contains the matching features and MNN coordinates are provided.
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| Submission date |
Feb 24, 2021 |
| Last update date |
Nov 15, 2023 |
| Contact name |
CHANGHEE LEE |
| E-mail(s) |
chlee@genetics.med.harvard.edu
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| Phone |
617-432-6534
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Lab |
Clifford Tabin
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| Street address |
77 AVENUE LOUIS PASTEUR
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| City |
BOSTON |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE167422 |
Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors [scRNA-seq] |
| GSE167423 |
Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors |
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| Relations |
| BioSample |
SAMN18051371 |
| SRA |
SRX10162930 |
| 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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