|
| Status |
Public on Oct 13, 2021 |
| Title |
CD8SP long-read [CD8SP_CCS] |
| Sample type |
SRA |
| |
|
| Source name |
CD8SP thymocytes
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: thymus
cell type: Sorted for mature CD8SP (CD8+CD4-TCRbeta+CD24-) from CD4 depleted samples
|
| Treatment protocol |
Two or three thymi were pooled for each sample and then depleted of CD4 expression cells (DP and CD4SP) or CD8 expressing cells (DP and CD8SP) using anti-CD4 or CD8 MACS magnetic beads (Miltenyi). The depleted samples were then sorted for mature CD4SP (CD4+CD8-TCRß+CD24-) or CD8SP (CD8+CD4-TCRß+CD24-) thymocytes on a FACS Aria Fusion (BD).
|
| Growth protocol |
C57BL/6 mice at 6-8-week of age were euthanised by asphyxiation with CO2 and the thymi were excised and placed in RPMI 1640 medium. Thymi were passed through a 100µm stainless steel mesh to produce single cell suspensions of thymocytes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was then extracted from the sorted cells with on GenElute Total RNA Purification columns (Sigma).
PacBio libraries were constructed following the IsoSeq Express Template Preparation for Sequel Systems protocol (www.pacb.com). For this procedure, first strand cDNA synthesis was performed using SMARTer PCR cDNA Synthesis Kit (Clontech), which also add necessary adaptor sequences for subsequent PCR amplification. PCR amplification of the cDNA was then performed using PrimerSTAR GXL DNA Polymerase (Clontech) and purified using AMPure PB magnetic beads (PacBio). The purified cDNAs were then end-repaired and ligated using the Template Prep Kit (PacBio) to generate the circular SMRTbell libraries. Each library was then sequenced on individual PacBio SMRT cells. For Illumina libraries, the RNA concentration and integrity of samples were first determined with a LabChip GX (Perkin Elmer). Paired-end RNA-seq libraries were then constructed with the TruSeq total RNA sample preparation kit with rRNA-depletion (Illumina) and sequenced on an Illumina NextSeq at the Ramaciotti Centre for Genomics, University of New South Wales.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
| |
|
| Data processing |
PacBio circular consensus sequencing data were processed using Iso-Seq SMRT Link (version 5.0.1.9585) to identify high-quality full-length sequences.
High-quality full-length sequences were mapped to mouse genome by GMAP (version2016-11-07). Cupcake ToFU (v9.0.2) was used to collapse redundant sequences and unify the CD4SP and CD8SP libraries.
SQANTI2 (version3.6) was used to perfom isoform quality assessment and annotation.
Filtered high-quality full-length PacBio transcripts were served as a reference.
FastQC was used to check Illumina short reads quality and Trimmomatic was used to trim and remove low quality reads.
RSEM (version 1.3.1) was used to align processed Illumina short reads to previously contructed full-length transcript reference.
SUPPA2 (version2.3) was used to identify alternative splicing patterns in full-length transcript reference and percentage of spliced-in (PSI) values were quantified using processed Illumina short reads.
Identifying splicing isoforms based on only Illumina data: Illumina short reads quaility was checked by FastQC and filtered and trimmed by Trimmomatic. Processed short reads were aligned to mouse reference by TopHat (v2.0.14) and then assembled by Cufflinks (v2.1.1). Two assemblies of CD4SP and CD8SP were compared by Cuffcompare.
Genome_build: GRCm38.97
Supplementary_files_format_and_content: PacBio long-read annotation information and normalised read counts. GTF files from Illumina short reads assemblies (including FPKM values)
|
| |
|
| Submission date |
Oct 09, 2020 |
| Last update date |
Oct 15, 2021 |
| Contact name |
Mark Chong |
| E-mail(s) |
mchong@svi.edu.au
|
| Phone |
61-3-92313444
|
| Organization name |
St Vincent's Institute of Medical Research
|
| Street address |
9 Princes Street
|
| City |
Fitzroy |
| State/province |
Victoria |
| ZIP/Postal code |
3065 |
| Country |
Australia |
| |
|
| Platform ID |
GPL24198 |
| Series (1) |
| GSE159290 |
A comparison of alternative mRNA splicing in the CD4 and CD8 T cell lineages |
|
| Relations |
| BioSample |
SAMN16403275 |
| SRA |
SRX9269763 |
