title: CT37A-DMSO_siNegative-H3K27me3
organism: Mus musculus
description: Protocols: Dissociate cells using Accutase treatment for 3-5min. Add ice cold PBS + 0.1% BSA and pipette up and down to obtain single cell suspension. Follow Cut&Tag protocol v2 (dx.doi.org/10.17504/protocols.io.z6hf9b6) Mouse embryonic stem cells were routinely cultured in 2i (1 μM PD0325901 [MEK(i)], 3 μM CHIR99021) or 2i with LIF (1:1000 LIF Qkine) following the protocols outlined in (Mulas et al., 2019). Cells were dissociated using Accutase (Millipore, SCR005) and pipetted to obtain a single cell suspension. The cell suspension was diluted in 5x wash buffer [DMEM/F12, 0.03% BSA Fraction V (Thermo Fisher)], span down and resuspended in fresh 2i or 2i/LIF medium before cell counting. Cells were plated at 1.5x10^4 cells/cm2. For maintenance, cells were plated on dishes coated with 0.1% gelatine for at least 15min at 37°C (Sigma-Aldrich, G1890), while all exit and differentiation experiments were performed in plates coated with 10μg/ml laminin in PBS for at least 30 min at 37°C (Sigma Millipore CC095). For all experiments, batch tested, home-made N2B27 was used [N2B27.BV, (Mulas et al., 2019). siRNA treatment and exit from naïve state All siRNA experiments were performed in 12 well plates in technical duplicates and biological triplicates, following the protocols indicated in (Mulas et al., 2019). For each well, we plated 60,000 cells, 20nM of total siRNA and 0.5μl Lipofectamine RNAiMax (Thermo Fisher) in 1ml 2i on laminin-coated plates. After overnight incubation (<18hrs), cells were either collected for RNA extraction and cDNA synthesis to determine the efficiency of knockdown or further differentiated. For differentiation experiments, cells were gently washed with 1ml PBS, before adding fresh media (e.g. N2B27 ± inhibitors). For each gene we used 2 siRNAs (10nM each). Samples were collected for Cut&Tag at 30hrs. siRNAs were purchased from Qiagen, Nanog siRNA: SI01323357, SI04460869; AllStars Negative Control siRNA: SI03650318). Recovery of labeled DNA ends with Phenol - Chloroform extraction. Concentration and clean up of PCR products with Ampure XP beads Nextera library preparation using NEBNext HiFi 2x PCR Master mix