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Sample GSM4065925

Query DataSets for GSM4065925

Status

Public on Apr 17, 2020

Title

WT4m2A

Sample type

SRA

Source name

Brain

Organism

Mus musculus

Characteristics

strain: C57BL/6
age: 4-month-old

Extracted molecule

total RNA

Extraction protocol

RNA was isolated from flow cytometry-sorted cell populations using RNeasy Micro Kit (74004, Qiagen)
A total amount of 10 ng RNA per sample was used as input material for the RNA sample preparations. Libraries were generated using SMART-Seq v4 Ultra Low Input RNA Kit (634892, Takara Bio USA, Mountain View, CA, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, first-strand cDNA synthesis from total RNA is primed by the 3’ SMART-Seq CDS Primer II A and uses the SMART-Seq v4 Oligonucleotide for template switching at the 5’ end of the transcript. PCR Primer II A amplifies cDNA from the SMART sequences introduced by 3’ SMART-Seq CDS Primer II A and the SMART-Seq v4 Oligonucleotide for 8 cycles. LD-PCR amplified cDNA is purified by immobilization on AMPure XP beads and then quantified with Agilent Bioanalyzer 2100 system. Approximately 200 pg was used for Nextera XT DNA Library Preparation Kit (Illumina, Cat. Nos. FC-131-1024 and FC-131-1096, San Diego, CA) to make cDNA libraries suitable for Illumina sequencing. Tagmented fragments were amplified for 12 cycles and dual indexes were added to each well to uniquely label each library. Concentrations were assessed with KAPA Library Quantification Kits (KK4844, KAPA, Biosystems, USA) and samples were diluted to ~2 nM and pooled. Pooled libraries were sequenced on the Illumina Novaseq platform and 150 bp paired-end reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: mm8
Supplementary_files_format_and_content: tab-delimited text files include readcount values for each Sample ...

Submission date

Sep 06, 2019

Last update date

Apr 17, 2020

Contact name

jie pan

E-mail(s)

panj@bjmu.edu.cn

Phone

18666886732

Organization name

Shenzhen Peking University – The Hong Kong University of Science and Technology Medical Center