GSM8777398: blood, time point 2, e397; Homo sapiens; RNA-Seq
Blood samples were collected in PAXgene RNA tubes (PreAnalytix, Switzerland), and leukocyte RNA was extracted from whole blood using the PAXgene Blood RNA Kit (Qiagen, Germany). RNA quality and quantity were assessed with the NanoDropTM 1000 Spectrophotometer (Thermo Fisher Scientific) and the Qubit 2.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific), respectively. DNA contamination was verified on a 1.5% agarose gel, and RNA integrity was evaluated using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent). Gene expression was measured using next-generation sequencing with the TruSeq® Targeted RNA Expression protocol (Illumina). Genes were selected based on four previous studies, two of them in the Brazilian High Risk Cohort for Mental Conditions (BHRCS). Briefly, 55 genes exhibited differential expression in the blood of BHRCS individuals who transitioned from low to high psychiatric symptoms over a 3- year follow-up period, indicating the onset of symptoms (10.1038/s41398-020-0772-3). Conversely, four genes were differentially expressed in individuals who showed a reduction in psychiatric symptoms, suggesting remission (10.1038/s41398-020-0772-3). Additionally, three genes displayed differential expression in the blood of BHRCS participants with major depressive disorder (10.1016/j.jpsychires.2017.03.015). Furthermore, we included 20 genes known to be differentially expressed in the brains of individuals with autism spectrum disorders, schizophrenia, bipolar disorder, depression, or general mental health conditions (10.1126/science.aad6469), as well as 6 endogenous control genes for blood samples for normalization procedures (10.1186/1755-8794-2-49). A total of 100 ng of RNA was reverse transcribed into cDNA using ProtoScript II Reverse Transcriptase (New England Biolabs). Libraries were prepared using the TruSeq Targeted RNA Expression Kit and the TruSeq® Targeted RNA Custom Oligonucleotide Pool (Illumina). Libraries were pooled and sequenced on the NextSeq 500 instrument (Illumina) using the MidOutput v2 kit (150 cycles) and 100 single end reads.