title: Illumina NovaSeq 6000 sequencing; RNA-seq experiment of mouse embryonic stem cells (mESCs), comparing TCF/LEF quadruple knock-out cells with wild type cells
library_construction_protocol: Cells collected from cell cultures, see growth protocol. mESCs were cultured in media containing N2B27 (gibco) supplemented with 2i/LIF, unless otherwise indicated, at the following final concentrations: PD0325901 (MEKi, Stem Cell), 1 µM; CHIR99021 (GSK3i, Stem Cell), 3 µM; LIF (Millipore), 1:1000. For passaging, cells were dissociated with TrypLE express (Gibco) for 3', pelleted by centrifugation (250g), and resuspended in fresh 2i/LIF. mES cells were maintained in filtered T25 flasks (TPP) coated with 0.1% Gelatine (Sigma-Aldrich, G1890). Cells were passaged every 2 days. Total RNAs from cells were extracted using standard Trizol protocol (Invitrogen), following the manufacture's instructions. Quality of isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Fragment Analyzer (Agilent, Santa Clara, California, USA). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq Stranded mRNA (Illumina, Inc, California, USA) was used in the succeeding steps. Total RNA samples (100-1000 ng) were polyA enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Fragment Analyzer (Agilent, Santa Clara, California, USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.