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Sample GSM6605146

Query DataSets for GSM6605146

Status

Public on Nov 30, 2023

Title

adult mouse anti-CD3 sorted splenocyte long-read PacBio scRNA-seq, replicate 3

Sample type

SRA

Source name

spleen

Organism

Mus musculus

Characteristics

tissue: spleen
cell type: mostly spleen T cells
age: 2 months
Sex: male
colony/strain: C57BL/6J

Extracted molecule

total RNA

Extraction protocol

Spleens were removed by dissection, transferred to a sterile petri dish containing PBS with 5% FBS, and minced using surgical blades. Spleen fragments were then ground through 100 μm and 40 μm cell strainers, pelleted, and resuspended in the ACK lysis buffer as described in the thymus tissue collection section. Cell density and viability were determined using Countness II Cell Counter. Subsequently, splenocytes were resuspended at 1 million cells per ml of 0.04% BSA/PBS. In order to sort out the splenocyte T-cell subsets for constructing 10× GEM single-cell libraries, splenocytes were resuspended at 1 million cells per ml of FACS buffer (PBS with 1% FBS and 5mM EDTA).splenocytes were pre-incubated with 1ug mouse Fc blocker (BD 553141) per 1 million cells/100ul FACS buffer for 5 minutes, and incubated with an anti-mouse CD3e (clone 17A2, eFlour450, 2 ug/ml) and an anti-mouse CD11b (M1/70, Alexa 488, 2 ug/ml) on ice for 40 minutes.after incubation, cells were washed and resuspended in the FACS buffer containing Sytox Blue Live/Dead dye for cell sorting using FACS BD Aria. Viable, single cells were gated, and the CD11b-/CD3e+ cells were collected for the generation of 10× GEM single-T-cell libraries
Biotinylated lockdown probes targeting all constant regions of the four TCR loci were added tp the 10× Chromium Next Gen Single-Cell 3’ V3 3′-barcoded scRNA-seq cDNA libraries using the xGen Hybridization and Wash kit with customized Discovery Pools from Integrated DNA Technologies, Inc (IDT, Coralville, Iowa).500 ng of amplified cDNA was mixed with the blocking probes and xGen human Cot DNA, and the mixtures were dried down in a SpeedVac system (ThermoFisher). The hybridization and washing steps were performed according to the protocol from IDT. The hybridization and post-capture amplification steps were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). Fourteen cycles were used for post-capture PCR amplification, and the amplified cDNA was evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip.
Amplified cDNA at >200ng per sample was used to generate Pacific Biosciences (PB) SMRTcell libraries. Full-length PB sequencing was performed on the PB Sequel II platform at Histogenetics (Ossining, NY)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Sequel II

Description

mostly TCR RNA
10x Genomics
mouse_spleen.TCRs_2m_3

Data processing

PacBio SMRT LINK V11 software to generate circular consensus sequences (CCS), requiring a predicted accuracy ≥ Q20.
mm10 + Gencode vM25 primary assembly annotation
demultiplex.<sample_name>.bam is a file containing the CCS demultipled reads

Submission date

Sep 28, 2022

Last update date

Nov 30, 2023

Contact name

Nimrod Daniel Rubinstein

E-mail(s)

[email protected]

Phone

9193082834

Organization name

Calico