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Sample GSM6359047

Query DataSets for GSM6359047

Status

Public on Oct 27, 2022

Title

CTLA4, scRNAseq

Sample type

SRA

Source name

Pancreatic Islet

Organism

Mus musculus

Characteristics

cell type: Pancreatic Islet
treatment: anti-CTLA-4
strain: NOD
age: 7 weeks

Treatment protocol

Approximately 7-week-old NOD mice were treated with anti-PD-L1 (Bio X Cell, Clone 10F.9G2) or anti-CTLA4 (Bio X Cell, Clone 9D9) every three days for 2 doses. Two days after the second dose mice were sacrificed and islets were harvested and processed into single cells. Samples from N=3 mice treated with anti-PD-L1 were combined and samples from N=3 mice treated with anti-CTLA-4 were combined.

Extracted molecule

total RNA

Extraction protocol

Single cell suspension in RT Master Mix was loaded on the Single Cell Chip and partition with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Upon dissolution of the Gel Beads in a GEM, the primers with the unique cell barcodes were released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produced barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture.
Full-length, barcoded cDNA was amplified by PCR to generate sufficient mass for library construction. Enzymatic Fragmentation and Size Selection are then used to optimize the cDNA amplicon size prior to library construction.
The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. Analysis steps such as demultiplexing, alignment, and gene counting, and visualization to generate expression data with single-cell resolution were performed.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

10x Genomics

Data processing

The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v4.0.0.
genome build: mm10
Supplementary files format and content: filtered_counts.h5ad is an AnnData object containing UMI counts for alpha, beta, and delta cells.

Submission date

Jul 20, 2022

Last update date

Oct 27, 2022

Contact name

Ana Luisa Perdigoto

E-mail(s)

ana.perdigoto@yale.edu

Organization name

Yale University

Street address

300 George Street