Agilent rna 6000 nano reagents port 1 kit

Total RNA was extracted from MDSC samples using TRI reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The quantity of total RNA was measured using an Agilent 2100 Bioanalyzer with Agilent RNA 6000 nano Reagents Port 1 kit (Agilent Technologies, Santa Clara, CA, USA). Optical density values at 260/280 were consistently above 1.9. The samples with intact, distinct ribosomal peaks were chosen for further analysis. The RNAs from three replicates were pooled as one RNA sample at each differentiation stage of MDSCs. Subsequently, the RNAs with low molecular weight were separated by 15 % PAGE, and the RNAs with molecules in the range of 18–30 nt were enriched and ligated with proprietary adapters to the 5' and 3' termini. A reverse transcription reaction followed by low-cycle PCR was performed to obtain sufficient product for high-throughput sequencing in Beijing Genomics Institute, China.

A and B were marked after collecting cell samples from CDMA and CDMB, respectively. RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, United States) and ribosomal RNA was removed with the mRNA-only kit (Epicenter Biotechnologies, Madison, WI, United States) according to the manufacturer’s instructions. RNA quality and quantity were determined with an Agilent RNA 6000 nano reagents port 1 kit (Agilent Technologies, Santa Clara, CA, United States) and an Agilent 2100 Bioanalyzer, respectively. The mRNA was separated from the total RNA using Sera-Mag Magnetic oligo(dT) particles (Sigma-Aldrich Corp., St. Louis, MO, United States) and chemically fragmented in mortar. The RNA was quantitated and reverse-transcribed into cDNA and the library was constructed and sequenced.