title: Illumina HiSeq 2500 sequencing; Bulk RNA-seq of cerebral organoids investigating the role of CHD8 in cortical development
design: Bulk RNA-seq of cerebral organoids investigating the role of CHD8 in cortical development
library_construction_protocol: Pools of three organoids at day 10 after Matrigel droplet embedding were dissociated in 700 µl Trizol (Invitrogen) by passing them 20-30 times through a 20G needle.  RNA extraction was performed by adding 140 µl chloroform (Sigma), followed by centrifugation for 15 min at 12000 g at 4 °C. The aqueous upper phase was transferred to a new tube and mixed with 1.5 volumes of 100% EtOH. RNA was extracted using Zymo-Spin™ IC columns (Zymo Research). In short, the aqueous phase/ethanol mixture was loaded onto the column, washed with 400 µl 70% EtOH before being treated with RQ1 DNAseI (Promega, 5 µl + 5 µl reaction buffer + 40 µl 70% EtOH) for 15 minutes at RT. After two washes with 70% EtOH the sample was eluted from the column with DEPC-treated H2O. RNA concentration was measured using the NanoDrop spectrophotometer (Thermo Scientific) and quality was assessed with the Bioanalyzer 2100 employing the RNA 6000 Nano kit (Agilent).  1 µg of RNA was used for cDNA library preparation using the QuantSeq 3' mRNA-Seq FWD Library Prep Kit (Lexogen) according to the manual. Library quality and size distribution were assessed with the High Sensitivity DNA Analysis Kit for the Bioanalyzer 2100 (Agilent). Concentration was measured on a Qubit® 2.0 fluorometer using the Qubit® dsDNA HS Assay Kit and libraries were pooled in equimolar amounts.