RNAseq of Homo Sapiens
We used Illumina TruSeq Stranded Total RNA Library Prep Gold (cat. no 20020599) to prepare the sequencing libraries from 250 ng of total RNA extracted from the Peripheral Blood Mononuclear Cells (PBMC) collected from patients at the three time points, as per manufacturers reference guide (1000000040499 v00). In summary, cytoplasmic and mitochondrial rRNA have been depleted using target-specific biotinylated oligos and Ribo-Zero rRNA removal beads, and pure RNA was fragmented using a divalent cation at a high temperature. Random primers and SuperScript IV reverse transcriptase were used to create the first strand cDNA from the fragmented RNA. Following RNaseH-mediated degradation of the RNA strand from the previous phase, DNA polymerase 1 was used to synthesize the second strand of cDNA. Adenylation of the double-stranded cDNA's 3' blunt end was done prior to indexing and amplification. AMPure XP (Beckman Coulter, A63881) was used for cleaning the final library. The library quality was checked using Agilent 2100 bioanalyzer followed by sequencing on NovaSeq 6000 using NovaSeq S2 v1.5 reagents with a 2x101 read length and 450 pM loading concentration.