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Sample GSM7291493

Query DataSets for GSM7291493

Status

Public on May 08, 2023

Title

NF1MEC2

Sample type

SRA

Source name

MECs

Organism

Rattus norvegicus

Characteristics

tissue: MECs
genotype: wildtype

Extracted molecule

total RNA

Extraction protocol

For RNA extraction of whole fat pads and tumors, fresh harvested samples were cut up and added to MPbio lysing matrix E tube. Cells were homogenized for 20 seconds in an MPbio homogenizer, then RNA was isolated from the supernatant using the Trizol-chloroform-isopropanol RNA extraction method. For RNA extraction of mature adipocytes, RNA was isolated using the Trizol-chloroform-isopropanol RNA extraction method. For RNA extraction of cultured epithelial cells and fibroblast, 1.2105 cells were plated into one well of a six-well plate. At 90-100% confluency, RNA was isolated using QIAGEN QIAshredder and RNeasy Kit manufacturer’s instructions.
Libraries were prepared by the Van Andel Institute Genomics Core from 500 ng of total RNA using the KAPA mRNA Hyperprep kit (v4.17) (Kapa Biosystems, Wilmington, MA USA). RNA was sheared to 300-400 bp. Prior to PCR amplification, cDNA fragments were ligated to IDT for Illumina TruSeq UD Indexed adapters (Illumina Inc, San Diego CA, USA). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems).Individually indexed libraries were pooled and 50 bp, paired-end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S2 sequencing kit (Illumina Inc., San Diego, CA, USA) to an average depth of 45M reads per sample. Base calling was done by Illumina RTA3 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

wildtype MECs

Data processing

Raw reads were trimmed of sequencing adapters with Trim_Galore
Trimmed reads were mapped to Rnor 6.0 assembly with STAR v2.5.2b using options ‘--twopassMode Basic’ and ‘--quantMode GeneCounts’ to directly output counts for all features from the ensemble annotation 6.0.90
Assembly: Rnor 6.0
Supplementary files format and content: raw_counts.tsv has the raw counts output from STAR mapping for features in the ensemble 6.0.90 annotation.

Submission date

May 03, 2023

Last update date

May 08, 2023

Contact name

Ian Beddows

Phone

000000000

Organization name

Van Andel Institute

Street address

333 Bostwick