title: Illumina HiSeq 2500 paired end sequencing; The extracellular function of LGALS3BP in neural progenitors during brain development
design: The extracellular function of LGALS3BP in neural progenitors during brain development
library_construction_protocol: Cerebral organoids were dissociated using a papain-based protocol (Miltenyi Biotec, Neural tissue dissociation kit (P)). Samples were selected at day 60 and processed enzymatically to get single-cell suspension. Firstly, each sample was cut into pieces and washed up to 3 times in 1x HBSS without Ca2+ and Mg2+ (HBSS w/o). Then samples were incubated for 15 min at 37C in the Enzyme Mix 1 with Papain (2 ml) and Enzyme Mix 2 (30 μl) was added once tissue started to disintegrate. In order to get sufficient dissociation, samples were gently triturated using 1 ml pipette tip and incubated for another 15 min at 37C. To generate single-cell suspension and remove clumps of cells, samples were applied to 30 and 20 μm diameter strainers and washed up to 3 times in HBSS w/o. Then samples were centrifuged at 300 RCF for 5 min and resuspended in HBSS w/o. Finally, a test of cell viability was performed using Trypan blue solution (0.4%) while counting cells by automatic cell counter (Countess, ThermoFisher). Single-cell suspension was used immediately for single-cell RNA-seq experiments (10X, Genomics, Chromium Single Cell 3' v2 Reagent Kit). iPSCs-derived cerebral organoids were generated as previously described (Lancaster and Knoblich, Nature Protocols, 2014). The iPSCs were reprogrammed from NuFF3-RQ human new-born foreskin feeder fibroblasts (GSC-3404, GlobalStem). Single stranded cDNA was generated in droplets according to 10X Genomics 3' Single cell Kit v2 guide instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay. cDNA libraries were prepared as described in the 10X Genomics user guide (Single Cell 3′ Reagent Kits User Guide PN-120233, 10X Genomics). Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.