GSM9669387: sputum_control_606; Homo sapiens; RNA-Seq
RNA was isolated from samples using the miRNeasy mini kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). Cells were lyzed in QIAzol lysis reagent and chloroform was added to perform a two-phase extraction. After centrifugation, the RNA in the aqueous phase was precipitated by ethanol and washed with the provided buffers from the kit using spin columns. Finally, it was eluted in 30 μL RNase-free water and the concentration was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Dreieich, Germany) and RNA integrity (RIN) was assessed using the Agilent RNA 6000 Nano Kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Carla, CA, USA). Rapid MACE-seq was used to prepare 3' RNA sequencing libraries. Samples of 100 ng of DNA-depleted RNA were used for library preparation, using the Rapid MACE-Seq kit (GenXPro GmbH, Germany). Fragmented RNA underwent reverse transcription using barcoded oligo(dT) primers containing TrueQuant unique molecular identifiers, followed by template switching. PCR amplified libraries were purified by solid phase reversible immobilization beads and subsequent sequencing was performed using the Illumina platform NextSeq 500