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Sample GSM5352832

Query DataSets for GSM5352832

Status

Public on Feb 18, 2022

Title

male brain replicate3

Sample type

SRA

Source name

male brain replicate3

Organism

Mus musculus

Characteristics

tissue: brain
development stage: Adult
gender: male
strain: C57BL/6J
library type: fr-firststrand (dUTP)

Extracted molecule

total RNA

Extraction protocol

Total RNAs from mouse (C57BL/6J) adult brain, cerebellum, heart, colon, and gonad in both sexes were extracted. The RNAs were classified into two parts, one part was subsequently treated with Ribo-off rRNA Depletion Kit to remove ribosome RNA (rRNA). Then the VAHTS TM Stranded mRNA-seq Library Prep Kit for Illumina was used for strand-specific library preparation. After finished, the library was suquenced on the Illumina Nova platform. The another part was used to constructe for cDNA using P Clontech SMARTer PCR cDNA Synthesis Kit and PrimeSTAR GXL DNA Polymerase, followed by library preparation by using SMRTbellTM Template prep and sequencing on the Pacific Biosciences Sequel I platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

71_clean

Data processing

RNA-Seq: High-throughput sequencing raw image data files were converted to reads using fqtools_plus analysis.
RNA-Seq: We used FastqC (0.11.8) to evaluate the raw reads quality. Sequencing report tool was MultiQC (0.9).
Iso-seq: The Raw data was subjected to SmrtLink Pipeline ccs to self-correct and obtain full-length circular consensus sequencing (CCS) reads based on PacBio recommended pipeline. We used the SmrtLink Pipeline classify subprogram to distinguish full-length non-chimeric sequences with the limitation of the transcript length longer than 300bp and applied the SmrtLink Pipeline cluster subprogram to cluster the full-length non-chimeric sequences into full-length transcripts, and retained the full-length transcript supported by at least one full-length non-chimeric sequences. The LoRDEC program was used to correct the full-length transcripts based on the results of Illumina RNA-seq to improve the accuracy of the third-generation transcripts.
RNA-Seq: Reads were mapped to the corresponding reference genomes (mm10, ENSEMBL98) by Hisat2 (2.0.5) with parameters “-x --rna-strandness RF” and “--known-splicesite-infile” followed by gene annotation that conbined reference annotation and Iso-seq full-length transcript.
RNA-Seq: The output of Hisat2 were converted to BAM format, sorted and only uniqe mapped reads were retained.
RNAseq: featurecounts (1.6.3) was used for counting reads with parameters "-s 2 -t exon -g gene_id".
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include raw read counts of all the genes for each RN-Aseq sample, as well as Iso-seq full transcript gtf files.
Supplementary_files_format_and_content: mouse_reference_iso.txt: conversion from PacBioID to geneID
Supplementary_files_format_and_content: mouse_final.gtf: full length transcripts
Supplementary_files_format_and_content: Count.matrix.txt: count matrix table

Submission date

Jun 02, 2021

Last update date

Mar 10, 2022

Contact name

Zhen-Xia Chen

E-mail(s)

zhen-xia.chen@mail.hzau.edu.cn

Organization name

Huazhong Agricultural University

Department

College of Life Science and Technology