Nanodrop nd 100 spectrometer

Cell pellets obtained after centrifugation of exponentially growing cultures were immediately re-suspended in 1 ml of 60°C hot TriReagent (Sigma-Aldrich, Steinheim, Germany) and transferred into a 2 ml cryovials containing acid washed glass beads (Sigma-Aldrich, Steinheim, Germany). As for DNA extraction, cell lysis was achieved with a Bio101 FastPrep instrument (Thermo Savant Illkirch, France) run at maximum speed (6.5 ms⁻¹) for 45 s. RNA isolation was then performed as described in [64 (link)]. In brief, after thawing the cell lysate on ice, 200 μl of pure chloroform was added to each sample. The sample was vortexed thoroughly and incubated for 10 min at room temperature. The aqueous phase was separated by centrifugation and transferred into a new vial together with 100% isopropanol and incubated at -20°C to precipitate the RNA. The pellet was collected by centrifugation at 4°C, washed with 70% ethanol, air-dried and re-suspended in RNase-free water (Qiagen, Hilden, Germany).
Only RNA samples with high quality (OD 260/280 > 2 and OD260/230 > 1.8), determined with a NanoDrop ND-100 spectrometer (PeqLab, Erlangen, Germany), and high RNA integrity, checked with the Agilent RNA Nano Chip Assay (Agilent, Santa Clara, USA), were used for transcriptome sequencing.

Total RNA was extracted from 2 × 10⁶ cultured primary human monocytes using the high pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. RNA preparations were then quantified by spectrophotometry (NanoDrop ND-100 Spectrometer, Peqlab, Erlangen, Germany) and equal amounts were reverse transcribed using Reverse Aid First Strand cDNA synthesis kits (Thermo Fischer Scientific, Karlsruhe, Germany). Obtained cDNA was used for quantitative PCR utilizing the “SYBR green ROX mix” (Thermo Fischer Scientific, Karlsruhe, Germany) and the following sequence-specific primers with regard to expression of protein coding genes: Actin fwd 5′-AGA GCT ACG AGC TGC CTG AC-3′, actin rev 5′- AGC ACT GTG TTG GCG TAC AG-3′, SOCS1 fwd 5′-TCC CCC TCA ACC CCG T-3′, SOCS1 rev 5′-CAT CCG CTC CCT CCA ACC-3′, IL-1β fwd 5′-AGC TGA TGG CCC TAA ACA GA-3′, IL-1β rev 5′-GCA TCT TCC TCA GCT TGT CC-3′.