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Sample GSM5074117

Query DataSets for GSM5074117

Status

Public on Feb 10, 2024

Title

Ly6C+_MHC2-_6h_rep3_L002

Sample type

SRA

Source name

WT 6h LyC6+ MHC2– rep3

Organism

Mus musculus

Characteristics

strain: C57Bl/6
tissue: Peritoneal Lavage
genotype: wildtype
Sex: Male
age: 8-12 weeks
treatment: i.p. Zymosan
time point: 6 hours
cell type: MoMac Ly6C+ MHCII-

Treatment protocol

Mice were treated with intraperitoneal injection of 200ug Zymosan A (Sigma)

Growth protocol

Parent animals were obtained from Jackson laboratories. Experimental mice were bred and housed in a specific pathogen free facility at National Jewish Health, Denver, CO, USA.

Extracted molecule

total RNA

Extraction protocol

Peritoneal lavage was collected using 8mL buffer (HBSS w/o Ca2+ Mg2+, 20mM HEPES, 1mM EDTA). Total lavage cells were stained with antibodies for FACS and sorted to purify monocytes/macrophages of the indicated phenotype. Total RNA was extracted using the Qiagen RNeasy micro kit according to manufacturer's instructions.
A Clontech SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Mountain View, CA, USA) and Nextera XT (San Diego, CA, USA) kit were used. Briefly, library construction started from isolation of total RNA species, followed by SMARTer 1st strand cDNA synthesis, full length dscDNA amplification by LD-PCR, followed by purification and validation. After that, the samples were taken to the Nextera XT protocol where the sample is simultaneously fragmented and tagged with adapters followed by a limited-cycle PCR that adds indexes. Once validated, the barcoded-pooled libraries were sequenced using 1×50bp chemistry on the HiSeq 2500 as routinely performed by the NJH Genomics Facility.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Ly6C+_MHC2-_6h_rep3_L002

Data processing

FASTQ files were generated using the Illumina bcl2fastq converter (version 2.17).
Nextera TruSight adapters were trimmed with skewer (version 0.2.2), which by default removes reads with a remaining length of less than 18 nt.
Trimmed reads were mapped with the STAR aligner (version 2.4.1d) to the GRCm38 assembly of the mouse genome using gene using gene annotations from Ensembl version 84.
Reads mapping to each gene of the Ensembl 84 annotation were counted with the featureCounts program from the Subread software package (v1.5.1).
Transcripts per million aligned fragments (TPM) were calculated from the gene counts using the sum of all bases covered by any transcript of a gene as its transcript length.
Genome_build: GRCm38
Supplementary_files_format_and_content: read counts and transcripts per million (TPM) assigned to each gene of the mouse Ensembl 84 annotation and sample, as tab-separated text tables: 1) output from subread featureCounts (2 subread_genecounts.txt* files), separated by sequencing run, 2) aggregated by adding different sequencing runs for each sample (counts and TPM)

Submission date

Feb 10, 2021

Last update date

Feb 10, 2024

Contact name

Donna Leslie Bratton

E-mail(s)

brattond@njhealth.org

Organization name

National Jewish Health

Department

Pediatrics