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Sample GSM5873148

Query DataSets for GSM5873148

Status

Public on Feb 11, 2022

Title

VM mouse #3

Sample type

SRA

Source name

Mouse lung FACS sorted for live CD45-EPCAM-PDGFRa-CD31+ endothelial cells

Organism

Mus musculus

Characteristics

cell type: Lung CD31+ endothelial cell
genotype: STING V154M mutant
tissue: lung

Extracted molecule

polyA RNA

Extraction protocol

Lungs from mice were dissected and digested using the GentleMACS lung digestion kit. Samples were then FACS sorted for live CD45-EPCAM-PDGFRa-CD31+ directly into RLT buffer. Limited RNA（more than 200pg, high-quality）was amplified with oligo-dT and dNTPs, incubated at 72℃and immediately put back on ice, then reverse transcribed to cDNA based on polyA tail. The template was switched to the 5' end of the RNA and the full-length cDNA was generated by PCR. Agilent 2100 bioanalyzer instrument (Thermo Fisher Scientific, MA, USA) was used to determine the average molecule length of PCR product. Purified cDNA from previous steps was fragmented into small pieces with fragment buffer by PCR, and the product was purified and selected by the Agencourt AMPure XP-Medium kit (Thermo Fisher Scientific, USA). cDNA was quantified by Agilent Technologies 2100 bioanalyzer.
Oligo(dT)-attached magnetic beads were used to purified 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 pair-end 100 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

samples processed with gentleMACS lung digestion kit
3_CD31_pos_A

Data processing

1. For Quality Control, we use FastQC to create qc outputs. There are optional read quality filtering (trimmomatic), read quality trimming (trimmomatic), adapter removal (cutadapt) processes available.
1. Bowtie2/Bowtie/STAR is used to count or filter out common RNAs (eg. rRNA, miRNA, tRNA, piRNA etc.).
1. RSEM is used to align RNA-Seq reads to a reference transcripts and estimates gene and isoform expression levels. Alternatively, Kallisto used for quantifying abundances of transcripts based on pseudoalignments.
1. HISAT2, STAR and Tophat2 are used to align RNA-Seq reads to a genome. Optionally, counting reads to genomic features such as genes, exons, promoters and genomic bins could be done by featureCounts.
1. Genome-wide Bam analysis is done by RseQC, Picard.
Genome_build: mm10
Supplementary_files_format_and_content: a csv file output by DESeq2 containing expected counts for detected genes, differentially expressed genes are tested for by false discovery rate

Submission date

Feb 08, 2022

Last update date

Jun 30, 2022

Contact name

Katherine A Fitzgerald

E-mail(s)

[email protected]

Phone

5088566518

Organization name

Umass Chan Medical