GSM8859516: B6 BMDMs Biological replicate 1; Mus musculus; RNA-Seq
BMDMs were lysed in TRIzol (Thermo Fisher Scientific; 15596026), and RNA was extracted using Qiagen RNeasy Mini Kit (74104) and RNase-free DNase Set (Qiagen; 79254), following the manufacturer's protocol. RNA purity and concentration was quantified using Nanodrop One (Thermo Fisher Scientific) and stored at -80 ºC. Total RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). Samples were initially treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contaminants. The next steps included performing rRNA depletion using QIAseq® FastSelect™−rRNA HMR kit (Qiagen, Germantown, MD, USA), which was conducted following the manufacturer's protocol. RNA sequencing libraries were generated with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs are fragmented for 15 minutes at 94°C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end-repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on one lane of a flow cell. After clustering, the flow cell was loaded on the Illumina HiSeq 4000 instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.