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Sample GSM3986288

Query DataSets for GSM3986288

Status

Public on Jul 28, 2021

Title

Vector-1

Sample type

SRA

Source name

primary neuron

Organism

Mus musculus

Characteristics

strain: C57BL/6
tissue: cortex
time: cultured day 10

Treatment protocol

Neurons were transduced with Vector or circScmh1 lentivirus.

Growth protocol

Primary neurons from C57BL/6J mice were isolated from embryonic day 14 (E14) mouse cortex. Neurons at 10 days were used in this study.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Agilent 2100 with RIN number >7.0.
Poly(A) RNA is purified from total RNA(5ug) using poly-T oligo-attached magnetic beads using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using divalent cations under high temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP. An A-base is then added to the blunt ends of each strand, then ligated to modified Illumina multiplex barcode adapters, which including custom Unique Molecular Identifiers for minimizing sequence-dependent bias and amplification noise according to (Shiroguchi et al. 2012), and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, The ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300 bp (±50 bp). At last, we performed the paired-end sequencing on an Illumina Novaseq TM 6000 following the vendor's recommended protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

A cDNA library constructed by technology from the pooled RNA from brain samples of mouse was sequenced run with Illumina sequence platform. Prior to assembly, the low quality reads(1,reads containing sequencing adaptors; 2,reads containing sequencing primer;3, nucleotide with q quality score lower than 20) were removed.
We aligned reads of samples to the UCSC (http://genome.ucsc.edu/) C57BL_6NJ mouse reference genome using HISAT package.
The mapped reads of each sample were assembled using StringTie. Then, all transcriptomes from Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. After the final transcriptome was generated, StringTie and edgeR was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample …

Submission date

Jul 30, 2019

Last update date

Jul 28, 2021

Contact name

Li Yang

E-mail(s)

220153005@seu.edu.cn

Organization name

Southeast University

Department

Pharmacology