 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 21, 2023 |
| Title |
ONT_T2_BR3 |
| Sample type |
SRA |
| |
|
| Source name |
spleen
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: spleen
cell type: transitional 2 B cells
Sex: female
flow cytometry_markers: CD19+ CD93+ IgM+ CD23+
|
| Treatment protocol |
B cell subsets were purified from 12-week old untreated mice
|
| Growth protocol |
Single cell suspensions were prepared from spleen by passing the organ through a 70mm cell strainer. B cells were pre-enriched through negative selection (Miltenyi B cell isolation kit, cat # 130-090-862) and T1, T2, MZ and FoB cell subsets FACS-sorted using anti-CD19 (6D5), anti-CD93 (AA4.1), anti-CD23 (B3B4), anti-CD21 (7G6) and anti-IgM (II/41) antibodies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Oxford Nanopore Technologies (ONT) sequencing libraries were prepared as described previously (Berrens et al, 2021, Nat. Biotech, DOI: 10.1038/s41587-021-01093-1; D'Angeli et al, 2022, Eur J Immunol, DOI: 10.1002/eji.202149781). Briefly, RNA was reverse transcribed using the Smart-seq2 protocol (Picelli et al, 2013, Nat Methods, DOI: 10.1038/nmeth.2639), cDNA was amplified using the KAPA HiFi Uracil+ hot start polymerase mix (Roche) and PCR products were purified using 0.6X AMPure XP beads (Beckman). Equal amount of cDNA libraries were pooled for a total of 200 fmol and sequenced with MinION R9.4.1 flow cell using the SQK-LSK109 kit on MinKNOW (21.02.1) according to manufacturers’ instructions.
Total RNA was extracted using RNeasy Mini Kit (Qiagen)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
| |
|
| Data processing |
Basecalling of reads was performed with guppy basecaller (4.0.11) in high accuracy mode. Reads with a mean sequence quality score higher than 7 were demultiplexed with the guppy_barcoder tool and processed with pychopper (2.5.0) in order to identify, orient and trim the full-length cDNA reads.
Pychopped reads were then aligned to mouse genome reference GRCm38 using minimap2 (2.17-r941) (Li et al, 2018, Bioinformatics, DOI: 10.1093/bioinformatics/bty191) with splice aware setting and analysed with FLAIR pipeline (Tang et al, 2020, Nat Commun, DOI: 10.1038/s41467-020-15171-6) for isoform identification.
Assembly: GRCm38
Supplementary files format and content: ONT_cDNAPCR_fullLength_counts.tsv: tab-delimited text file containing gene names and IDs together with the number of full length transcript isoforms detected for each gene in each sample, from FLAIR analysis.
|
| |
|
| Submission date |
Oct 24, 2022 |
| Last update date |
Jun 21, 2023 |
| Contact name |
Louise Matheson |
| E-mail(s) |
[email protected]
|
| Organization name |
The Babraham Institute
|
| Department |
Laboratory of Lymphocyte Signalling and Development
|
| Street address |
Babraham Research Campus
|
| City |
Cambridge |
| ZIP/Postal code |
CB22 3AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24973 |
| Series (1) |
| GSE178728 |
An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells |
|
| Relations |
| BioSample |
SAMN31430852 |
| SRA |
SRX18006170 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
|
|
|
|
 |
