|
| Status |
Public on Sep 13, 2019 |
| Title |
Knee-joint at E14.5 single-cell RNAseq by 10X Genomics |
| Sample type |
SRA |
| |
|
| Source name |
Lgr5-EGFP-IRES-creERT2/+ heterozygous embryos, GFP expressed in Lgr5+ cells in the forming knee joint
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Lgr5-EGFP-IRES-creERT2/+ heterozygous
cell type: GFP expressed in Lgr5+ cells in the forming knee joint
developmental stage: E14.5
strain background: C57bl/6
|
| Growth protocol |
Lgr5GFP/+ (c57bl6) mice were crossed with c57bl6 wt mice to generate the embryos
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissue was digested with 0.2% Collagenase II, 0.1% Dispase and 0.1% Hyaluronidase. Cells were washed twice with HBSS, and filtered through 40um cell strainer twice before loading onto the 10x genomics machine.
Single cell encapsulation and cDNA libraries were prepared by Chromium™ Single Cell 3’ Reagent Kits v2 and Chromium™ Single Cell A Chip Kit. Cells in suspension are counted and loaded into individual wells of 10X Chromium Single Cell chip. Single cells are then encapsulated into Gel in-beads emulsions (GEM) by 10X Chromium Controller. Single Cell 3’ Reagent Kits v2 is used to perform downstream steps, in brief as follows: reverse transcription is performed on GEMs, followed by cDNA clean up and amplification. The double-stranded cDNA will then go through enzymatic fragmentation, adapter ligation, index PCR and SPRIselect size selection per manufacturer’s protocol. Library size and concentration is determined by Qubit and Bioanalyzer assays. The libraries are denatured and diluted to optimal concentration. Cluster generation is performed on the flow cell with HiSeq PE Cluster Kit v4 by cbot. Illumina HiSeq SBS Kit v4 is used for sequencing run at 100 bp for read 1 and read 2.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Description |
Knee-joint at E14.5 single-cell RNAseq by 10X Genomics.
|
| Data processing |
Using software from Illumina (bcl2fastq), sequencing reads were assigned into individual samples. An average of 72% of the bases achieved a quality score1 of Q30 where Q30 denotes the accuracy of a base call to be 99.9%.
The demultiplexed FASTQ files were then processed with cellranger-2.1.0 (10X Genomics, Inc), a proprietary pipeline that perform alignment, quantification, and light-weight analyses in one go.
R1 contains barcode (bp 1-16) and UMI (bp 11-26).
Genome_build: Grcm38(mm10)
Supplementary_files_format_and_content: space-delimited text files containing gene expression table of the cells in sparse format (gene-id, cell-id, number-of-UMIs)
barcodes.tsv: cell barcodes for cells that pass non-background cutoff
genes.tsv: a list of all genes
matrix.mtx: gene expression table in sparse format
|
| |
|
| Submission date |
May 08, 2019 |
| Last update date |
Sep 14, 2019 |
| Contact name |
Peikai Chen |
| E-mail(s) |
pkchen1@hku.hk
|
| Phone |
852-258315217
|
| Organization name |
The University of Hong Kong
|
| Department |
Faculty of Medicine
|
| Street address |
Sassoon Road 5
|
| City |
Pok Fu Lam |
| ZIP/Postal code |
10000 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL18480 |
| Series (1) |
| GSE130919 |
Lgr5 and Col22a1 mark progenitor cells in the lineage toward juvenile articular chondrocytes |
|
| Relations |
| BioSample |
SAMN11605332 |
| SRA |
SRX5807396 |
