title: GSM8369973: Neutrophils (mpx+), 8 hours post-injury, DMSO, biol rep 2; Danio rerio; RNA-Seq
library_construction_protocol: Trunks containing the lesion site and same region of age-matched uninjured controls were dissociated in a solution containing 2.5 mg/ml collagenase IV, 15 mM HEPES, 25 mM D-Glucose, 2% goat serum in HBSS without magnesium and calcium, incubated at 28.5ºC shaking at 300 rpm, and vigorously dissociated using a pipette. Next they were filtered and stained with DAPI (1ul/ml) for 10 min at 4C. Single-cell suspensions were used to sort GFP+ cells directly into TRIzol using a FACSAria III (BD Biosciences). All Samples were processed in less than two hours. Bulk RNA Sequencing was performed at Single Cell Discoveries. Total RNA was extracted from 2500-4800 GFP+ cells using the standard TRIzol protocol. mRNA was processed following an adapted version of CEL-seq. Samples were barcoded with CEL-seq primers during the reverse transcription and pooled after second strand synthesis TruSeq small RNA primers (Illumina)