title: GSM5639693: PMN-DAMPs-JLRNASEQ-22; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were lysed in RiboZol (Amresco) and kept at -80℃ until use. Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen), following the procedures described by the manufacturer Library was prepared using 25 ng RNA input and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). First, ribosomal RNA was removed using the NEBNext rRNA Depletion Kit v2 (Human/Mouse/Rat)(NEB) as indicated by the manufacturer's instructions. Briefly, samples with a RIN between 2 and 6 were fragmented for 8 min at 94°C whereas samples with a RIN ≥7 were fragmented for 15 min at 94°C for a target insert size of 200 bp. Next, the first and the second cDNA strand were synthesized, followed by an Ampure XP bead (Beckman Coulter) clean up. Then the samples were end-prepped, and the adaptor was ligated. Adaptor was diluted following the manufacturer's instructions. The adaptor-ligated samples were purified with Ampure XP beads before being amplified. For the PCR enrichment, a different Unique Dual Index Primer Pair (NEB) was added to each sample and 14 cycles were used. A last purification using Ampure XP beads was done to obtain the final library. If needed, libraries were pooled at the same concentration and the last bead purification was done a second time to remove adapter-dimer. The quality of the library was assessed as follows; size distribution was determined with the D1000 assay on the Tapestation (Agilent). The dsDNA HS assay from Qubit (ThermoFisher Scientific was used to evaluate the concentration.