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Sample GSM5105266

Query DataSets for GSM5105266

Status

Public on Feb 25, 2021

Title

Epididyaml primary adipocytes - Prmt1 FKO_rep2

Sample type

SRA

Source name

Epididyaml primary adipocytes

Organism

Mus musculus

Characteristics

strain: C57BL/6N
cell type: Epididyaml primary adipocytes
genotype: Prmt1 adipocyte-specific KO

Extracted molecule

total RNA

Extraction protocol

Total RNA concentration was calculated by Quant-IT RiboGreen(Invitrogen, #R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA screentape(Agilent). Only high-quality RNA preparations, with RIN greater than 7.0, were used for RNA library construction.
A library was prepared with 1ug of total RNA for each sample by Illumina TruSeq mRNA Sample Prep kit (Illumina, Inc., San Diego, CA, USA). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T oligo‐attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature.
The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase(Invitrogen) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the indexing adapters. The products are then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany). Indexed libraries were then sequenced using the HiSeq4000 platform (Illumina,San Diego, USA) by the Macrogen Incorporated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

KO-8

Data processing

We preprocessed the raw reads from the sequencer to remove low quality and adapter sequence before analysis and aligned the processed reads to the Mus musculus (mm10) using HISAT v2.1.0
HISAT utilizes two types of indexes for alignment (a global, whole-genome index and tens of thousands of small local indexes). These two types’ indexes are constructed using the same BWT (Burrows–Wheeler transform)/ a graph FM index (GFM) as Bowtie2.
The reference genome sequence of Mus musculus (mm10) and annotation data were downloaded from the NCBI. And then, transcript assembly of known transcripts was processed by StringTie v1.3.4d.
Expression abundance of transcript and gene were calculated as read count or FPKM value (Fragments Per Kilobase of exon per Million fragments mapped) per sample.
Genome_build: mm10

Submission date

Feb 24, 2021

Last update date

Feb 25, 2021

Contact name

Seung-Hoi Koo

E-mail(s)

koohoi@korea.ac.kr

Organization name

Korea University

Department

Life Sciences