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Sample GSM2750965

Query DataSets for GSM2750965

Status

Public on Aug 24, 2017

Title

DMSO-rep3

Sample type

SRA

Source name

PC-12 cell line

Organism

Rattus norvegicus

Characteristics

passages: 10-20
differentiation: NGF-differentiated

Treatment protocol

For stimulation of cannabinoid receptors 1 and 2, the full CB1/CB2 receptor agonist WIN 55,212-2 mesylate (Tocris; Tocris Bioscience, Minneapolis, MN, USA) was used at 5μM. For stimulation of Dopamine 1 (D1) receptors, a D1 agonist was used (SKF 81297 hydrobromide; Tocris) for ~30 minutes at 1μM. All drugs were dissolved in dimethyl sulfoxide (DMSO).

Growth protocol

PC12 cells were maintained in BioCoat™ Collagen IV 75cm² culture flasks (Corning; Corning Inc., Corning, NY, USA) to facilitate attachment and were incubated at 37°C in an atmosphere containing 5% CO2. During maintenance, PC12 cells were grown in 1X cellgro™ RPMI 1640 medium with L-Glutamine (Corning), supplemented with 10% horse serum (SIGMA), 5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 1X Pen Strep Glutamine (Gibco). All drug-treatment experiments, unless otherwise noted, were performed using NGF-differentiated PC12 cells, of passage (P) ~10-20, plated on BioCoat™ Collagen IV multiwell plates (Corning). For differentiation, PC12 cells were maintained in 1X cellgro™ RPMI 1640 medium with L-Glutamine (Corning), supplemented with 1% horse serum (SIGMA), 1X Pen Strep Glutamine (Gibco) and 100ng/ml of recombinant murine β-NGF (Peprotech; PeproTech Inc., Rocky Hill, NJ, USA), for seven days according to the literature.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated using the Direct-zol™ RNA MiniPrep Plus w/ TRI Reagent® kit
KAPA Stranded RNA-Seq Kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

QD56-3

Data processing

Sequencing was performed on a HiSeq 3000/HiSeq 4000 System (Illumina; Illumina Inc., San Diego, CA, USA) for a paired-end 150 bp run. Data quality check was done on the Sequencing Analysis Viewer (SAV, Illumina). Demultiplexing was performed with the bcl2fastq2 v 2.17 program (Illumina).
Reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Gene expression levels were normalized to total counts.
Differentially expressed genes were identified using the differential gene expression (GSA) algorithm (Partek; Partek Inc., St. Louis, MO, USA).
Genes showing altered expression with FDR < 0.05 and more than 2-fold changes were considered differentially expressed (Table S2; first worksheet).
Genome_build: RGSC 6.0/ rn6
Supplementary_files_format_and_content: tab-delimited text file includes normalized counts for each sample

Submission date

Aug 22, 2017

Last update date

May 15, 2019

Contact name

Philippe Melas

Organization name

Columbia University

Lab

Kandel's lab

Street address

1051 Riverside Drive