title: NextSeq 500 sequencing; Transcriptome analysis of neurospheres derived from Cstb-KO mouse embryo brains during differentiation
design: Transcriptome analysis of neurospheres derived from Cstb-KO mouse embryo brains during differentiation
library_construction_protocol: CSTB-deficient mice (Cstb−/−) were obtained from The Jackson Laboratory (129-Cstbtm1Rm/SvJ; stock no. #003486). Wild-type mice of the same age and background were used as controls. The research protocols were approved by the Animal Ethics Committee of the State Provincial Office of Southern Finland (decision ESAVI/10765/04.10.07/2015 ).The ganglionic eminences (MGE+LGE region) of E13.5 mouse embryos were dissected and mechanically dissociated to obtain a single cell suspension. Cells were plated onto 60mm non-treated culture dishes at a density of 2x10^5 cells/ml in 5ml maintenance medium, consisting on Dulbecco's modified Eagle's media (DMEM; Lonza) and F12 (Gibco) 3:1 supplemented with 2% B-27 , EGF (Millipore) at 20 ng/ml and Murine FGF-basic (PeproTech Inc.) at 40 ng/ml (Ciccolini and Svendsen 1998). After 6 days in culture at 37°C, ripe primary neurospheres were dissociated with TrypLE Express Enzyme (Gibco) and sub-cultured in maintenance media at a density of 6x10^4 cells/ml. After 4 days, ripe secondary neurospheres were either dissociated and sub-cultured or plated for differentiation. For differentiation, cells were plated onto poly-ornithine-coated surfaces at a density of 3x10^4 cells/cm2 in differentiation media (Standard: Neurobasal media (Gibco) supplemented with 2% B-27 and 2% Fetal bovine serum (FBS; Biowest). Single secondary neurospheres of ≈200 μm of diameter (≈400 cells) were manually collected and either lysed directly (Day 0 samples) or plated for differentiation in round-bottom 96-well plates and lysed 1, 5 or 12 days after differentiation. Lysis buffer contained Tris pH7, 10mM EDTA, Proteinase K and 1%w/v SDS. Cell lysis was followed by 30-minute incubation at 50°C and RNA purification through RNA clean & concentrator kit (Zymo). Each experimental situation was represented by six colonies derived from three different embryo pools. 2 µl of lysate was used to make 48-plex RNA-seq libraries, which were prepared using modified STRT method with unique molecular identifiers (UMIs). (Islam et al. 2011 and Islam et al. 2014). In order to remove amplification artefacts at high copy numbers, we have used longer UMIs of 8 bp.