title: GSM6735817: larvae, cold dissociated, rep 2; Danio rerio; RNA-Seq
library_construction_protocol: 10  anesthetized larvae were washed twice in ice cold HBSS (Life Technologies GmbH #14175095) and resuspended in 200µl psychrophilic protease mix (1x Native Bacillus licheniformis Protease (Creative Enzymes #NATE-0633) in HBSS, 10 mM 4sU) and incubated on ice for 30 min. Dissociation was aided by pipetting up and down with a 200 µl tip ~25 times every 5 minutes. Enzymes were inactivated by adding 15 µl 10% BSA  (Sigma-Aldrich; #A4503) before spinning down and resuspending the cells in TRIzol RNA was extracted using TRIzol-Chloroform-Isopropanol according to the manufacturer's instructions. Iodiacetamide Treatment was carried out on extracted RNA in 50% DMSO as described in Herzog et. Al 2017 (https://doi.org/10.1038/nmeth.4435). libraries for RNA-seq were prepared following a CELseq protocol as published in Holler & Junker 2019 (https://doi.org/10.1007/978-1-4939-9009-2_9). Sequencing adapters were switched – Insert is in read 1!