|
| Status |
Public on Jun 21, 2023 |
| Title |
E10.5a TEPC, scRNAseq SmartSeq2 |
| Sample type |
SRA |
| |
|
| Source name |
thymus
|
| Organism |
Mus musculus |
| Characteristics |
tissue: thymus
cell type: thymic epithelial progenitors
genotype: wild type
developmental stage: E10.5 primordia
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Individual EPCAM+PLET1+ cells from E10.5 or E12.5 primordia were sorted into 96-well BD Precise WTA Single Cell Encoding Plates. The plates were sealed and stored at -80oC until library preparation. Four plates (376 cells) were collected per timepoint.
Reverse transcription was performed in the presence of ERCC RNA control. The 96 samples from each plate were then pooled and purified with Agencourt® AMPure® XP magnetic beads (clean-up step). Second strand synthesis was carried out and the resulting products cleaned up. Following adaptor ligation and clean-up, ligation products were PCR amplified for 18 cycles overnight, and all subsequent steps were performed in a separate post-amplification area. The amplified products were cleaned up and quantified using Qubit dsDNA HS Assay. 50ng of the products then underwent random primer extension and 12 cycles of further amplification. The amplified libraries were cleaned up and quantified with Qubit assay. The libraries were stored at -80oC until sequencing. The libraries were sequenced on Illumina MiSeq at the Wellcome Centre for Human Genetics in Oxford within 6 months of preparation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Preprocessing of the FASTQ files was performed using the BD: Precise Whole Transcriptome Assay Analysis Pipeline v2.0. This pipeline performs all required steps to demultiplex, align, and quantitate sequencing reads from BD™ Precise Whole Transcriptome Assay. After performing a quality control assessment using FastQC, the pipeline filters and demultiplexes sequencing reads based on the sequence of the 8 base sample barcode. It aligns reads from each sample to the transcriptome using STAR, with feature counting by the union rules of HTSeq-count. The initial quantification is performed by counting the unique molecular indices (MI) mapped to each feature. Lastly, the pipeline uses two error correction algorithms to remove MI errors. In brief, MI errors that are derived from sequencing base calls and PCR substitution errors are identified and adjusted to a single MI barcode using Recursive Substitution Error Correction™ (RSEC). Subsequently, MI errors that are derived from library preparation steps or sequencing base deletion errors are adjusted using Distribution-Based Error Correction™ (DBEC).
Assembly: GRCm38.p5
Supplementary files format and content: 7Bridges PDF Summary Report = Provides an overview of each sequenced library.
Supplementary files format and content: 7Bridges DBEC Molecular Index Mapping report = Reports the number of unique molecular indices seen associated with each individual transcript per well.
Supplementary files format and content: 7Bridges DBEC Read Mapping report = Reports the number of reads that mapped to a given transcript in each of 96 wells.
Supplementary files format and content: 7Bridges Sample Barcodes (Sample Index) report = Summarizes the number of reads that have been assigned to each well.
Supplementary files format and content: 7Bridges Raw Molecular index counts = Reports the raw counts for each unique molecular index detected for each gene in each well.
Supplementary files format and content: 7Bridges Raw read counts = Reports raw read counts for each gene in each well.
Supplementary files format and content: 7Bridges FastQC Reports = Provides metrics to assess overall quality of the input sequencing reads.
Supplementary files format and content: 7Bridges RSEC Molecular Index Counts = Reports RSEC adjusted MI counts for each gene in each well.
Supplementary files format and content: 7Bridges Status Report = Reports sequencing depth metrics according to gene and well. The summary is in the MI_Detection column in the DBEC Molecular Index Mapping report.
|
| |
|
| Submission date |
May 17, 2023 |
| Last update date |
Jul 14, 2023 |
| Contact name |
Catherine Clare Blackburn |
| E-mail(s) |
c.blackburn@ed.ac.uk
|
| Phone |
+447810223009
|
| Organization name |
University of Edinburgh
|
| Department |
School of Biological Sciences
|
| Lab |
Thymus generation and regeneration
|
| Street address |
IRR North, 5 Little France Drive
|
| City |
Edinburgh |
| ZIP/Postal code |
EH16 4UU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE232765 |
Thymic epithelial cell potency and fate in early organogenesis assessed by single cell transcriptional and functional analysis [Smart-Seq2] |
|
| Relations |
| BioSample |
SAMN35148844 |
| SRA |
SRX20405360 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7373475_458648_SampleIndex.csv.gz |
1.3 Kb |
(ftp)(http) |
CSV |
| GSM7373475_DBEC_MolecularIndexMapping_458648.csv.gz |
549.2 Kb |
(ftp)(http) |
CSV |
| GSM7373475_DBEC_ReadMapping_458648.csv.gz |
653.9 Kb |
(ftp)(http) |
CSV |
| GSM7373475_RSEC_MolecularIndexCounts_458648.csv.gz |
578.1 Kb |
(ftp)(http) |
CSV |
| GSM7373475_StatusReport_458648.csv.gz |
844.1 Kb |
(ftp)(http) |
CSV |
| GSM7373475_WTA_SummaryReport_458648.pdf |
1.2 Mb |
(ftp)(http) |
PDF |
| GSM7373475_WTCHG_458648_1_fastqc.b64html.txt.gz |
244.8 Kb |
(ftp)(http) |
TXT |
| GSM7373475_WTCHG_458648_1_fastqc.zip |
383.8 Kb |
(ftp)(http) |
ZIP |
| GSM7373475_WTCHG_458648_2_fastqc.b64html.txt.gz |
275.1 Kb |
(ftp)(http) |
TXT |
| GSM7373475_WTCHG_458648_2_fastqc.zip |
436.4 Kb |
(ftp)(http) |
ZIP |
| GSM7373475_raw_MolecularIndexCounts_458648.csv.gz |
588.8 Kb |
(ftp)(http) |
CSV |
| GSM7373475_raw_ReadCounts_458648.csv.gz |
712.5 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
