title: GSM4776967: 10_moderate_TreatmentStage [lnc_16]; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh peripheral blood by Ficoll-Paque (GE Healthcare) density gradient centrifugation, and were stored at -80°C until the use for total RNA extraction. Total RNA was extracted by using the RNeasy kit (TianGen, Beijing, China) according to the manufacturer's instructions. The purity, concentration, and integrity of total RNA were checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA), the Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, USA), and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 System (Agilent Technologies, CA, USA), respectively. Besides, RNA degradation and contamination was monitored on 1% agarose gels. A total amount of 1μg total RNA per sample was used to prepare for the rRNA-depleted cDNA library. Ribosomal RNA was removed by Epicentre Ribo-zeroTM rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, sequencing libraries were generated using the rRNA-depleted RNA by NEBNext UltraTM Directional RNA Library Prep Kit for Illumina (NEB, USA) and sequenced on an Illumina HiSeq 4000 platform to generate 150bp paired-end reads. For small RNA library, a total amount of 2μg total RNA per sample was used as the input material. Sequencing libraries were generated using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following manufacturer's recommendations. Small RNA libraries were sequenced on an Illumina Hiseq 2500 platform and 50bp single-end reads were generated.