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Sample GSM6674466

Query DataSets for GSM6674466

Status

Public on Jun 21, 2023

Title

ONT_FoB_BR4

Sample type

SRA

Source name

spleen

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: spleen
cell type: follicular B cells
Sex: male
flow cytometry_markers: CD19+ CD93- CD21- 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