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Sample GSM7146951

Query DataSets for GSM7146951

Status

Public on Mar 27, 2024

Title

WT rep1

Sample type

SRA

Source name

bone marrow B220+ cells

Organism

Mus musculus

Characteristics

cell type: bone marrow B220+ cells
genotype: WT

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the TRIzol reagent. RNA quality and quantity were determined using a Nano Drop and Agilent 2100 bioanalyzer.
Oligo(dT)-attached magnetic beads were used to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. Afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on DNBSEQ platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format.
The clean reads were mapped to the reference genome using HISAT2 (v2.0.4).
Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then expression level of gene was calculated by RSEM (v1.2.12).
Differential expression was performed using DESeq2 (version 1.4.5).
Assembly: GCF_000001635.26_GRCm38.p6
Supplementary files format and content: WT_and_PELI2_CKO_genes.fpkm.anno.txt: Tab-delimited text file includes FPKM values for each Sample.

Submission date

Apr 04, 2023

Last update date

Mar 27, 2024

Contact name

Yan Xu

E-mail(s)

xy18260090078@outlook.com

Organization name

Shandong University

Street address

No. 44 Wenhua West Road