title: mRNA-seq of H3.3 mutant (K9A; K27A; K79A) and control mESCs
design: mRNA-seq of H3.3 mutant (K9A; K27A; K79A) and control mESCs
library_construction_protocol: Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. RNA was extracted from ~10 million cells using the RNeasy Mini kit (Qiagen) and DNase digestion was performed using TURBO DNase (Thermo Fisher Scientific), according to the manufacturer's instructions and an additional clean-up of the RNA was then performed using the RNeasy Mini kit (Qiagen). Quality of the RNA was assessed on Bioanalyzer using the Agilent RNA 6000 Nano Kit and samples with a RNA integrity number (RIN) of 10 were used for library generation. For each sample, 100 ng of total RNA was used as input for mRNA selection and conversion to cDNA using the NEBNext Poly(A) Magnetic Isolation Module (New England BioLabs). Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Preparation Kit for Illumina (New England BioLabs), according to the manufacturer's instructions. After Qubit quantification, the quality of individual libraries was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.