title: GSM9444954: [HEK293] [Cycloheximide]; Homo sapiens; OTHER
library_construction_protocol: Cells were extracted with TRIzol™ (as per manufacter's instructions) and further cleaned with Zymo® Research Clean & Concentrator-5 kit, as per manufacturer's instructions. The probed RNA (0,5-1 µg) was reverse transcribed using TGIRT-III™ Reverse transcriptase. Briefly, to 5 μL of probed RNA, 1 μL of 5X Reverse Transcription buffer (250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2), 0.5 μL of random hexamers (10 μM) and 0.5 μL of dNTPs (10 mM) was added. The mixture was incubated for 8 min at 98 °C, to simultaneously fragment and denature the RNA, and immediately transferred to ice. Sample was supplemented with 0.25 μL of SUPERaseIn (20 U/μL), 0.25 μL DTT (0.1 M) and 0.25 μL TGIRT-III™ Reverse transcriptase (200 U/L). Reverse transcription was carried out at typical conditions (25 °C 10 min, 57 °C 2 hours, hold 4 °C). Then, 1 μL of ice-cold proteinase K (1 μg/μL) was added to each sample, to degrade the reverse-transcriptase. Reaction was carried out at 37 °C for 20 minutes. Proteinase K was deactivated by adding 1 μL proteinase inhibitor cocktail (Sigma Aldrich, cat. P186; 1:2 dilution in water). Library was then prepared using the of NEBNext® Ultra II™ workflow.