GSE202312
mRNA transcriptome sequencing of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) from Lyz2-Cre;Trpv2fl/fl and Trpv2fl/fl mice.
Femurs and tibias were dissected from Trpv2fl/fl and Lyz2-Cre;Trpv2fl/fl mice. Bone marrow was flushed from the bones, and the red blood cells were lysed with ACK lysis buffer. Debris was removed by passing cells through a 70 µM strainer and cells were counted via Countess II FL Automated Cell Counter. Approximately 5 × 106 bone marrow cells were cultured in 100 mm dishes in DMEM (Thermo Fisher Scientific, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% streptomycin-penicillin at 37 °C in a humidified incubator gassed with 5% CO2. GM-CSF (20?ng/mL, Peprotech) and M-CSF (10?ng/mL, Peprotech) were added to the bone marrow cultures for differentiation of BMDCs and BMDMs, respectively. The medium was changed every 3 days. On day 7, cells were collected and homogenized in 2 ml of TRIzol (Invitrogen). Total RNAs were prepared and the quality of RNAs was determined by agarose gel electrophoresis and spectrophotometer analysis. TruSeq Stranded mRNA LT Sample Prep Kit was used to synthesized the first strand cDNA through the action of random primers and reverse transcriptase, the second strand cDNA is synthesized with the first strand cDNA as the template, cDNA library construction followed by sequencing on an Illumina NovaSeq 6000 platform. RNA-sequencing reads were first trimmed to remove poly(A) and unqualified reads with cutadapt, then aligned to Mus musculus GRCm38 genome with Ensembl. The numbers of counts were summarized at the gene level using HTseq.  Gene expression values were computed from fragments per kilo bases per million fragments (FPKM) values produced by addition of a pseudocount of 1 and log2 transformation of the results. Overall design: BMDCs from Trpv2fl/fl mice (n=2) and Lyz2-Cre;Trpv2fl/fl mice (n=2) were used,respectively. BMDMs from Trpv2fl/fl mice (n=2) and Lyz2-Cre;Trpv2fl/fl mice (n=2) were used,respectively.