RNA seq of Homo Sapiens: non-mining region resident male PBMCs
PBMCs were isolated from venous whole blood on Histopaque gradient, washed, and stored in RNAlater (Sigma-Aldrich, Darmstadt, Germany) at -40°C. PBMCs collection for RNA extraction was done at the same time as blood plasma samples collection for analysis of metal levels. RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN, Germantown, Maryland, USA) according to the manufacturer's instructions. Poly-A selected strand-specific mRNA library was prepared with Illumina Stranded mRNA Prep kit (Illumina, San Diego, California, USA). The quality and concentration of both RNA samples and libraries were checked before downstream processing on the Agilent 2100 Bioanalyzer with appropriate kits. The sequencing was performed on the Illumina NovaSeq-6000 platform with 38.5 million single-end 100 bp reads on average generated per sample. The runs and sequencing quality were within normal ranges, i.e., Q30% greater than 90%.   Quality checking of the sequencing data was performed using Illumina Basespace reports and the program FastQC (V0.10.1). Reads were aligned against the human genome hg38 using STAR (version 2.7.1a) with the parameters "--outSAMtype BAM SortedByCoordinate' and "--quantMode GeneCounts TranscriptomeSAM" specified but otherwise default parameters. Adapter sequences were not trimmed. The median unique alignment rate was 89.53% (+/- 2.84%). For estimating cell type composition, TPM values were calculated using RSEM with the GFF file GCF_000001405.40_GRCh38.p14_genomic.gff and the parameter "--trusted-sources BestRefSeq".