title: GSM7875170: Brain 10 - diencephalon, scRNAseq; Danio rerio; RNA-Seq
library_name: GSM7875170
library_construction_protocol: Adult zebrafish brain tissue was extracted, optionally dissected to select a specific region of interest (telencephalon, diencephalon, mesencephalon or rhombencephalon) and then dissociated with a trypsin protocol. Briefly, the samples were incubated in 750 µl of HBSS-glucose solution and dissocated with 15 µl of 0.05% trypsin-EDTA (Gibco) for 30 minutes at 37°C with intermittent mixing. The disociation was stopped by addition of 750 µl of a BSA-EBSS-HEPES solution. Further sample handling steps were carried out on ice and for centrifugation at 4°C. The sample was filtered through a 100 µm filter, washed with cold HBSS and resuspended in 100-200 µl HBSS with 0.05% BSA, then filtered through a 35 µm filter and loaded on the 10x Chromium Controller. For the scSLAM-seq samples, a papain dissociation protocol (Worthington kit, according to manufecturer's instructions) was used followed by an adapted scSLAM-seq protocol for labeling nascent transcripts (see sample-specific entry). The standard transcriptome libraries were constructed according to the manufacturer's protocol (10X Genomics). For targeted libraries of scars used for lineage tracing, the cDNA obtained during the 10x protocol was used to set up individual PCR reactions for each target, specifically amplifying the sgRNA target site. The libraries for targeted PCR were constructed with primers compatible with the 10X Genomics kit, so they could be sequenced on the same platform.