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| Status |
Public on Nov 30, 2023 |
| Title |
ETV2plus NCs, scRNAseq |
| Sample type |
SRA |
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| Source name |
hiPSC H9
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| Organism |
Homo sapiens |
| Characteristics |
cell line: hiPSC H9
treatment: vascularized
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| Extracted molecule |
total RNA |
| Extraction protocol |
For nuclei isolation, all following steps were performed on ice with pre-cooled buffers and centrifugation steps were performed at 4 oC. In brief, the snap-frozen samples were thawed using 500 µl ice-cold homogenization buffer [HB; consisting of 320 mM sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Tris pH 7.8, 0.1 mM EDTA, 0.1% NP40, 1X complete protease inhibitor (Roche), 1 mM β-mercaptoethanol and RNase-In inhibitor (Promega)] for 5 min to prevent the nuclei from rupturing. The samples were then homogenized with pestle A (5 gentle strokes) and pestle B (15 gentle strokes) and incubated on ice for 5 min. The homogenates were filtered using FlowmiTM 70 µm cell strainers and the filtrates were centrifuged at 500g for 5 min. The resulting pellets were resuspended in 2.65 ml washing buffer (HB without NP40) and 2.65 ml gradient medium (5 mM CaCl2, 50% Optiprep, 3 mM magnesium acetate, 10 mM Tris pH 7.8, 1X complete protease inhibitor, 1 mM β-mercaptoethanol and RNase-In inhibitor) and the resulting solution was added gently on top of a cushion solution (29% Optiprep, 150 mM KCl, 30 mM MgCl2, 60 mM Tris pH 7.8 and 250 mM sucrose) for centrifugation at 7700 rpm for 30 min at 4 oC. The nuclei pellets were resuspended in 25 µl resuspension buffer (1X PBS, 2% BSA), filtered using FlowmiTM 40 µm cell strainers and performed nuclei counting using LUNA automated cell counter (Logos Biosystems) according to the manufacturer’s instructions
The diluted nuclei suspensions were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) (Next GEM Single Cell 3’ library and Gel Bead Kit v3.1) according to the manufacturer’s protocol (10x User Guide; CG000204, Revision D). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all performed according to the manufacturer’s protocol. Individual sample quality was assessed using a Tapestation (Agilent). Qubit 2.0 (ThermoFisher Scientific) was used for library quantification before pooling libraries. The final library pool was sequenced on NextSeq2000 (Illumina) instrument using NextSeq 1000/2000 P3 kit v3 for 2 lanes of 100-base-pair paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
10x Genomics
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| Data processing |
The Cell Ranger version 6.1.1 was used to process, align, and summarize unique molecular identifier (UMI) counts against the 10X Genomics pre-built human GRCh38 reference genome datasets (2020-A, July 7, 2020). Each sample was processed using the semi-supervised machine learning classifier – Debris Identification using Expectation Maximization (DIEM) to eliminate debris and empty droplets. More specifically, Debris and test sets were specified for droplets using set debris_test_set(sce, min_counts = 200, min_genes = 100) and genes with no expression were filtered out with filter_genes(sce, cpm_tresh = 10). Cell type clustering was initialized by performing PCA analysis using get_pcs(sce, n_var_genes = 2000, min_genes = 200, n_pcs = 30, threads = 8, seedn = 1234) and k-means algorithm using init(sce, k_init = 20, nstart_init = 30, min_size_init = 10, threads = 8). We run expectation maximization algorithm run_em(sce, threads = 8, model = ”DM”) to estimate the parameters of the multinomial mixture model and assign clusters for each droplet in the test set using assign_clusers(sce). We removed clusters that were assigned as debris using the command function call_targets(sce, clusters = "debris", thresh_score = NULL, min_genes = 0).
Assembly: GRCh38
Supplementary files format and content: rds
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| Submission date |
May 30, 2023 |
| Last update date |
Nov 30, 2023 |
| Contact name |
Yoke Chin Chai |
| E-mail(s) |
yoke.chin.chai@imec.be
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| Organization name |
KU Leuven
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| Department |
Development & Regeneration
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| Lab |
Stem Cell Institute Leuven
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| Street address |
Herestraat 49, ON5
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| City |
Leuven |
| State/province |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL30173 |
| Series (1) |
| GSE233741 |
Gene expression profile at single nucleus level of the generated neural concentroids with or without vascularization |
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| Relations |
| BioSample |
SAMN35533794 |
| SRA |
SRX20543095 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7433714_SKT016.01scRNAseq_YC_ETV2Plus_DIEM.rds.gz |
41.2 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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