title: GSM8895366: Monocytes, LPS, 1,25D-primed, RNA replicate 2; Homo sapiens; RNA-Seq
library_name: GSM8895366
library_construction_protocol: RNA-seq: cells were preserved in TRIzol (1 ml per 5 × 10⁶ cells; Zymo Research) at -80 °C overnight before RNA extraction. Total RNA was isolated using the High Pure RNA Isolation Kit (Roche) with minor modifications. Chloroform (1:5 ratio) was added to thawed TRIzol samples to induce phase separation. Phase-lock gel heavy tubes (5Prime) were used to efficiently separate proteins into the organic phase, DNA at the interface, and RNA in the aqueous phase. RNA quality and quantity were assessed using a Bioanalyzer (Agilent 2100; RNA Nano 6000 Assay), ensuring a RIN (RNA integrity number) > 8. Ribosomal RNA (rRNA) was depleted using the NEBNext rRNA Depletion Kit, and complementary DNA (cDNA) libraries were prepared from the rRNA-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, following the manufacturer's protocol. Library quality was confirmed using the Agilent DNA 1000 Chip, and indexed libraries were pooled prior to sequencing. RNA-seq libraries were sequenced on an Illumina NextSeq500 system (Illumina) at 75 bp read length using standard protocols at the Gene Core facility of the EMBL (Heidelberg, Germany).