Early transcriptomic response of innate immune cells  to subcutaneous BCG vaccination of mice
Bacille Calmette-Guerin (BCG) vaccination might contribute to nonspecific enhancement of resistance to various infections. Thus, BCG in addition to the formation of specific immunity against mycobacteria is also capable of inducing “trained immunity” - the ability of the innate immune system to maintain the memory of past infections and use it to develop immune defenses against new ones. In this study, we performed RNA sequencing of innate immune cells derived from mice bone marrow and spleen three day after subcutaneous BCG vaccination to evaluate early transcriptomic response of innate immune cells Overall design: We performed RNA sequencing of innate immune cells derived from murine bone marrow and spleen three day after subcutaneous BCG vaccination. To do this, we obtained single-cell suspensions from the organs, stained the suspensions with conjugated anti-CD45, anti-CD11b, anti-NK1.1, anti-Ly6G, and anti-Ly6C antibodies, and subjected the suspen-sions to fluorescence-activated cell sorting (FACS). The prepared cell suspensions were used for FACS sorting and three cell populations were collected: monocytes [CD45+CD11b+Ly6C+], neutrophils [CD45+CD11b+Ly6G+] and NK-cells [CD45+CD11b-NK1.1+]. The samples were collected for each mouse from both control and BCG vaccinated groups. Monocytes and neutrophils were collected during sorting of bone marrow cell suspensions and NK-cells were collected from spleens. Then we have pre-pared double-indexed cDNA libraries for Illumina sequencing from the collected samples. The quality of the cDNA libraries was confirmed via Agilent Tape Station 2200 system, and the resulting cDNA library mix was subjected to NovaSeq 6000 sequencing. The RNA sequencing data was further processed with HISAT2 (GRCm38/mm10 was used as the reference genome), featureCount (GENCODE vM23, GRCm38.p6 was used as the reference transcriptome) and DESeq2 tools.