GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3746308

Query DataSets for GSM3746308

Status

Public on May 01, 2020

Title

ctrl3

Sample type

SRA

Source name

control_FACS sorted pancreatic beta-cells

Organism

Rattus norvegicus

Characteristics

strain background: Sprague-Dawley
age: adult
genotype/variation: Control
cell type: FACS sorted pancreatic beta-cells

Treatment protocol

Cell transfection– Dispersed rat islet cells or FACS-sorted β-cells were transfected with a pool of 4 siRNAs directed against rat Scrt1 or negative control (On-Target plus #081299-02 and #001810-10 respectively, Dharmacon) using Lipofectamine RNAiMAX (Thermofisher). RNA extraction was performed 48h after transfection.

Growth protocol

Islet isolation, dispersion and cell sorting: Islets were isolated by collagenase digestion of the pancreas (Gotoh et al., 1987) followed by Histopaque density gradient and hand-picking to separate them from digested exocrine tissue. At the end of the isolation, islets were incubated for 2h in RPMI 1640 GlutaMAXTM medium (Invitrogen) containing 11 mM glucose and 2 mM L-glutamine and supplemented with 10% fetal calf serum (Gibco), 10 mM Hepes pH 7.4, 1 mM sodium pyruvate, 100 µg/mL streptomycin and 100 IU/mL penicillin. Dissociated islet cells were obtained by incubating the islets in Ca2+/Mg2+ free phosphate buffered saline, 3 mM EGTA and 0.002% trypsin for 5 min at 37°C. For some experiments, rat islet cells were separated by Fluorescence-Activated Cell Sorting (FACS) based on β-cell autofluorescence, as previously described (Guay et al., 2019; Kohler et al., 2012). Sorted islet cells were seeded on plastic dishes coated with extracellular matrix secreted by 804 G rat bladder cancer cells (804 G ECM) (Parnaud et al., 2008). Enrichment of α - and β-cells was evaluated by double immunofluorescence staining using polyclonal guinea pig anti-insulin (dilution 1:40, PA1-26938 Invitrogen) and polyclonal mouse anti-glucagon (dilution 1:1000, Abcam Ab10988), followed by goat anti-guinea pig Alexa-Fluor-488 and goat anti-mouse Alexa-Fluor-555 (diluted 1:400, Thermofisher A11073 and A21422, respectively) secondary antibodies. On average, β-cell fractions contained 99.1 ± 0.9% insulin-positive cells and 0.6 ± 0.6% glucagon-positive cells and α-cell-enriched fractions contained 10.6 ± 8.2% insulin-positive cells and 88.8 ± 8.2% glucagon-positive cells.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using miRNeasy micro kit (Qiagen) followed by DNase treatment (Promega).
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Quality control of the sequencing was performed with FastQC
Sequencing adaptors were removed using trimmomatic
An index of Rn6 cDNA was build for Kallisto
alignment of reads was performed using Kallisto
Subsequent analysis were performed with sleuth (differential expression analysis) and cluster profiller (annotation enrichment analysis)
Genome_build: Rno6
Supplementary_files_format_and_content: annotated count table with est counts, TPM, transcripts ID, gene ID,gene name, for each sample.

Submission date

May 02, 2019

Last update date

May 01, 2020

Contact name

Jonathan Aryeh Sobel

E-mail(s)

jonathan.sobel@weizmann.ac.il

Phone

00972587889058

Organization name

Weizmann Institute of science