GSM5034621: CTI 41; Homo sapiens; RNA-Seq
Human blood samples collected were immediately mixed with RNALater (Invitrogen, Carlsbad, CA) and kept frozen until used for RNA isolation. Total RNA was isolated from 2ml blood sample (0.5 ml blood and 1.5 ml RNALater) each using Ribopure Blood Kit (ThermoFisher Scientific, Waltham, MA), including DNA treatment. The RNA extracted was quantified using a Nanodrop ND-1000 (ThermoFisher Scientific) and quality assessed via an Agilent 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA) using Agilent RNA 6000 Nano Reagents. Only RNA samples with an RNA Integrity Number (RIN) >8.0 were used to construct next generation sequence (NGS) libraries. Immediately prior to NGS library synthesis, 1.4 mg total RNA per blood sample was used to deplete alpha and beta globin mRNA, using the GLOBINClear kit (ThermoFisher Scientific, Waltham, MA). 0.5mg globin-depleted RNA was then used as input for the Illumina TruSeq Stranded mRNA Kit to synthesize NGS libraries. Library synthesis was carried out using the default protocol in the Low Sample TruSeq Stranded mRNA Sample Preparation Guide with the following two modifications: fragmentation time was reduced to 3 minutes to achieve insert lengths ranging between 125 and 250 bp and the PCR amplification was reduced to 12 cycles. Individual cDNA libraries were quantified using a Qubit 3.0 Fluorometer (Invitrogen by ThermoFisher Scientific) and the dsDNA HS Assay Kit (Invitrogen by ThermoFisher Scientific). The average fragment size for each library was determined using an Agilent 2100 Bioanalyzer with High Sensitivity DNA Reagents. Libraries were used in 2 x 125 basepair sequencing using the Illumina HiSeq 2500 (Illumina, San Diego, CA) in high output mode with v4 HiSeq Cluster and SBS Kits (Illumina).
GEO Accession
GSM5034621