Bioanalyser high sensitivity

DNA was extracted from cells sorted from single retinas. 50–100 ng of DNA was used as an input for bisulfite conversion (Zymo Gold Kit). The converted DNA was used to prepare whole genome bisulfite libraries using Illumina Truseq DNA methylation preparation kit (EGMK81312) following manufacturer recommendation. PCR product was purified using AMPureXP beads (Beckman Coulter—A63880) and controlled on Bioanalyser High sensitivity (Agilent 5067-4626). The samples were run on an Illumina HiSeq2500 generating 100 bp paired-end reads (rapid-run).

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.