|
| Status |
Public on Dec 31, 2022 |
| Title |
young mouse 3 ( 8 weeks) [Sequel] |
| Sample type |
SRA |
| |
|
| Source name |
bone marrow cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Lineage negative, cKit (Cd117) positive (LK) cell fraction
strain: C57BL/6
Sex: female
age: 8 weeks
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bone marrow was isolated from spine, femora, tibiae, and ilia,blood cell depletion was performed with ammonium chloride lysis and lineage negative cells were isolated using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit
3’ end single cell RNA-Seq using the 10X Genomics Chromium (V2 Kit) according to manufacturer’s protocol
Libraries compatible for Illumina platform and for Pacific Biosciences Sequel/Sequel II systems were prepared from input cDNA
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
| |
|
| Description |
single cell fulllenght cDNA (10x Genomics)
|
| Data processing |
Illumina data
Raw Illumina sequencing data were analysed with the 10X Genomics CellRanger pipeline (version 3.0.2) to obtain a single-cell expression matrix object.
Using Seurat v3 in R data were normalised using NormalizeData
data from multiple samples were merged using the FindIntegrationAnchors and IntegrateData commands
count matrices produced by short-read sequencing for individual libraries were combined in Seurat using FindIntegrationAnchors and IntegrateData functions
Illumina and PacBio reads were integrated in Seurat using the CreateAssayObject command to add the long-read data to an existing Seurat object already containing the short-read data
Pacbio data
Circular Consensus reads (CCS) were generated using smrtlink-6.0.0.47841 amnd the command "ccs --numThreads=24 --noPolish --minLength=50 --maxLength=15000 --minPasses=1 --minPredictedAccuracy=0.8"
Reads with identified polydA or polydT were demultiplexed using bbduk and the 10X genomics barcodes identified from the short-read analysis
Long-reads were mapped to the mouse genome (mm10) using Minimap 2 (v2.17), and to the gencode (vM19) transcriptome. De novo transcript annotation was performed with TALON v.5 was used to generate custom transcriptome using gencode v.M24 and GRCm38.p6. Minimum identity threshold 0.9, coverage 0.8. Novel features filtered with N=5 reads in K=3 samples. Transcriptome then filtered for mis-called antisense transcripts. Novel features were merged with v.M24 reference to make custom annotation. Count matrices were generated using HTSeq, the alignments to the genome (GRCm38.p6, Minimap2) and the TALON annotation
Novel exons were identified by investigating the alignment of the reads to the transcriptome
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: gene level counts
Supplementary_files_format_and_content: de novo annotation
Supplementary_files_format_and_content: gene levels counts
|
| |
|
| Submission date |
Feb 12, 2021 |
| Last update date |
Dec 31, 2022 |
| Contact name |
Laura mincarelli |
| E-mail(s) |
laura.mincarelli@earlham.ac.uk
|
| Organization name |
Earlham Institute
|
| Street address |
Norwich Research Park, Colney Lane, Colney
|
| City |
NORWICH |
| State/province |
County (optional) |
| ZIP/Postal code |
NR4 7UG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24198 |
| Series (1) |
| GSE166709 |
Integrated long- and short-read single-cell RNA-sequencing |
|
| Relations |
| BioSample |
SAMN17296743 |
| SRA |
SRX9837722 |
