title: GSM8687146: Cther, lib697, 60C, Mock, RTHIV, rep1; Acetivibrio thermocellus; RNA-Seq
library_name: GSM8687146
library_construction_protocol: The aqueous phase was collected after centrifugation and mixed with an equal volume of ethanol. RNA was processed with the NEB Monarch Total RNA Miniprep Kit. DNase digestion to eliminate genomic DNA. Remaining primers left in the reaction were digested by addition of 3 μL ExoSAP-IT (Applied Biosystems, 78205) to the 20 μL reverse transcription reaction, and incubation at 37°C for 12 min. RNA was degraded by adding 2.5 μL of NaOH 1M and 1 μL of 0.5M EDTA, and incubation at 70°C for 12 min. The base was neutralized with the addition of 1M HCl, cDNA was cleaned using MyOne silane beads. An adapter was added to the 3' end of the cDNA by ligating 50 pmoles 5iLL-22 DNA adapter using T4 RNA Ligase 1, High concentration (NEB, M0437M), DMSO 4%, ATP 1mM and PEG8000 25% at 23°C for 3 h. After clean-up with MyOne silane beads, we amplified the cDNA library for 12 cycles using Illumina Library PCR primers F and R and KAPA HiFi HotStart ReadyMix (Roche, KK2601). Amplified libraries were cleaned with Kapa pure beads (Roche, KK8002), quantified using Qubit (Life Technologies) and the distribution of library size was determined using TapeStation (Agilent Technologies).