Illumina HiSeq 2500 sequencing; Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb
Effect of rabies virus infection on gene expression in GABAergic neurons in the Zebrafish olfactory bulb
EnvA-RV-GFP was injected to olfactory bulb in Tg[gad1b:Gal4, UAS:TVA-mCherry] fish. Fish were kept in the standard fish system at 36 degree for 3-4 days. Afterwards, olfactory bulbs were extracted from 3-5 fish and the samples were pooled. After standard dissociation processes, cells were sorted by their fluorescent markers using LSRII. Batch 1 (pool 1 to 3) was done 7 days before batch 2 (pool 4 to 8). RNA was purified using single-cell RNA purification Kit (Norgen, cat. 51800) mRNA-seq libraries were generated using the SmartSeq2 approach (Picelli et al, Nature protocol 2014), with the following modifications: For cDNA pre-amplification, up to 10ng of RNA was used as input (typically 1-3ng), and Reverse Transcription was performed using Superscript IV (Thermo Fisher Scientific - 50C for 10min, 80C for 10min). Amplified cDNA (1ng) were converted to indexed sequencing libraries by tagmentation, using in-house purified Tn5 (Picelli et al, Genome Research 2014) and Illumina Nextera primers.