GSM7850088: Late CMDM_02; Mus musculus; RNA-Seq
On day 5 post-challenge, mice ears were cut into small pieces by razors, followed by tissue dissociation by using Multi Tissue Dissociation Kit 1 and gentleMACS Octo Dissociator with Heaters according to the protocol for the program named 37C_WSDK_2. Cells were then subjected to FACS sorting and four Mo-Mac subpopulations (Ly6ChiPD-L2lo, Ly6ChiPD-L2hi, Ly6CloPD-L2hi and Ly6CloPD-L2lo) were isolated. Total RNA was lysed from 5,000-80,000 isolated Mo-Mac cells by using lithium dodecyl sulfate-based lysis/storage buffer. mRNA was isolated from total RNA by Dynabeads M-270 streptavidin with biotin-oligo (dT)25 (Thermo Fisher Scientific). First- and second-strand cDNA synthesis was performed by Superscript reverse transcriptase IV (Thermo Fisher Scientific) and Kapa HiFi DNA polymerase (Roche). Whole-transcriptome library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, catalog #: E7805). Ligated products were purified and performed the barcoding PCR. Pooled libraries were sequenced by Illumina Novaseq 6000 sequencer (Illumina) and NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles) (Illumina).