Ter51020

For each condition 20 10^ˆ6 of Triptolide treated Drosophila cells were spiked with 2 10^ˆ6 untreated mouse embryonic stem cells. scRNA protocol was adapted from (Nechaev et al., 2010 (link)). Cells were re-suspended in ice-cold lysis buffer (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10min on ice, span down. Nuclei were washed with ice cold (10mM Tris (pH = 7.4), 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and nuclear pellets were dissolved in Trizol (Thermofisher). RNA was size selected (17-200bp) using a two-step column purification strategy (RNA clean and concentrator, Zymo-R1016). 10 μg of purified RNA was successively treated by 5′ dephosphorylation - 20U at 37°C for 30min (Epicenter - RP8092H); 5′ terminator exonuclease - 1U in Buffer A at 30°C for 60min (Epicenter - TER51020); cap-clip decapping enzyme – 5U at 37°C for 90min. After each reaction, short RNA was column purified (RNA clean and concentrator, Zymo-R1016). The resulting RNA was used for library preparation using TruSeq small RNA library (Illumina). Libraries were purified on 6% TBE gels (150-300bp – Novex - EC6265BOX). Size distribution of the libraries were controlled on Bioanalyser High sensitivity (Agilent 5067-4626). Two biologically independent inhibition time courses were performed. The samples were run on an Illumina NextSeq generating 38bp paired-end reads.