Illumina HiSeq 2500 sequencing; Single-cell transcriptome of cerebral organoids with and without methanol fixation
Single-cell transcriptome of cerebral organoids with and without methanol fixation
Single cell suspensions from organoids were obtained by Papain based dissociation (Miltenyi Biotec, Neural tissue dissociation kit (P)) using whole organoids. Cell suspension of each organoid was split into two aliquots. Cells in one aliquot per line were pooled. Methanol fixation was applied to cells in the remaining aliquots, following the same procedure as described previously (Alles et al. 2017). Afterwards, the fixed cells were rehydrated following the procedure described (Alles et al. 2017). Single cells were encapsulated along with beads and reverse transcription reagents into droplets using 10X microfluidic chip system. Reverse transcription of first strand cDNA was performed in droplets. Induced pluripotent stem cells were maintained in feeder-free culture on matrigel coated plates. Cerebral organoids were generated according to the protocol by Lancaster et al. (Nature Protocols, 2014).  Single stranded cDNA was cleaned up and amplified after breaking the droplets generated during the sample collection using the 10X 3' Single cell Kit v2 according to the manufacturer's instructions. Quality and quantity of the resulting cDNA was assessed using Agilent's Bioanalyzer High Sensitivity DNA assay.  Libraries were constructed following the 10X library construction protocol v2. Briefly, cDNA was fragmented, end repaired and adaptor sequences were ligated, followed by a PCR to itroduce library specific sequencing primers as provided by 10X. Libraries were quantified and checked for size distribution using Agilent Bioanalyzer DNA High Sensitivity Assays.
Experimental Factor: fixation
fixed