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Sample GSM6909390

Query DataSets for GSM6909390

Status

Public on Mar 31, 2023

Title

Sample 3 - UT, 3h, rep3

Sample type

SRA

Source name

Bone marrow-derived macrophages

Organism

Mus musculus

Characteristics

cell type: Bone marrow-derived macrophages
genotype: wild-type
treatment: untreated
time: 3h

Treatment protocol

For RNA Sequencing, 1 Million BMDMs per condition were left untreated (control), stimulated with 30 µM S14 or 100 ng/mL LPS for 3 h or 18 h before harvesting cells for RNA isolation.

Growth protocol

Bone marrow-derived macrophages (BMDMs) were generated by culturing bone marrow cells in DMEM supplemented with 10 % FCS, 1 % Penicillin/Streptomycin and 15 % L929 cell-conditioned medium (LCM). On day 6 of differentiation, BMDMs were collected and plated for experiments. Experiments were performed in DMEM supplemented with 10 % FCS and 1 % Penicillin/Streptomycin, unless stated otherwise.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using the RNeasy Mini Kit together with the RNase-free DNase Set for DNA digestion (both Qiagen) according to manufacturer instructions
Libraries were prepared manually according to the manufacturer’s instructions using QuantSeq 3' mRNA-Seq FWD Library Prep Kit. Briefly, 150 ng total RNA was used as input for reverse transcription with oligo(dT) primer containing an Illumina-compatible sequence at its 5’ end. After RNA removal, the second strand of the cDNA was synthesized by a random primer containing an Illumina-compatible linker sequence at its 5’ end. Finally, individual sample barcodes (dual indexing) for multiplexing were introduced via 15 cycles of PCR (determined by qPCR). All libraries were analyzed for adapter dimers, size distribution and concentration on a Fragment Analyzer (Agilent). After pooling the libraries in an equimolar ratio, the concentration and the size distribution of the lane mix was analyzed by Qubit (Thermo Fisher Scientific) and by Fragment Analyzer (Agilent), respectively. A 2 nM dilution of the lane mix was denatured and diluted for sequencing on a NextSeq 2000 instrument

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

Matrix Column S

Data processing

Using cutadapt version 1.18 (https://cutadapt.readthedocs.io/en/stable/), the reads of the sequencing run were scanned for adapter contaminations, continuous polyA sequences and continuous polyG sequences at the 3’ end and had the contaminations removed if they were found. The reads of the samples prior to adapter trimming and after adapter trimming were analyzed with FastQC version v0.11.7 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
The reads were aligned to the spike-in complemented Ensembl release 101 of the Mus musculus high coverage assembly GRCm38 from the Genome Reference Consortium. The alignment was performed with the splice-aware aligner STAR version 2.6.1a (https://github.com/alexdobin/STAR)1. The alignments were quantified based on the annotations of Ensembl GRCm38.101 and the spike-in specific annotations of Lexogen with the featureCounts software program version 1.6.4 of the subread analysis package (http://subread.sourceforge.net/).
A differential gene expression analysis was conducted using DESeq2 (version v1.18.1; https://bioconductor.org/packages/release/bioc/html/DESeq2.html ). The analysis used the counts of unique alignments. Significance was determined at adj P<0.1.
Assembly: GRCm38
Supplementary files format and content: tab-separated values (tsv) file including library names, gene IDs and individual normalized counts

Submission date

Dec 29, 2022

Last update date

Mar 31, 2023

Contact name

Friederike Sophie Gorki

Organization name

Institute of Innate Immunity

Street address

Venusberg-Campus 1

City

Bonn