title: GSM6735854: sc_DR_2; Danio rerio; RNA-Seq
library_construction_protocol: 10 dechorionated larvae were anaesthetized by placing them in 2 ml centrifuge tubes on ice for 5 minutes. We then washed them twice using ice cold HBSS (Life Technologies GmbH #14175095) and removed the supernatant afterwards. For dissociation the larvae were resuspended in 500 µl TryplE Mix (1xTrypleE (Life Technologies GmbH #A1217701) in HBSS, 1mM EDTA pH 8 (Molekula #900493), 10 mM 4sU) and incubated at 37°C for 30 min. When all visible tissue had disappeared, we inactivated the enzymes by adding 15 µl 10% BSA  (Sigma-Aldrich; #A4503) to the suspension and spun down at 1000 g for 5 minutes (4 °C). Finally, the cells were washed once in ice cold HBSS (no 4sU added). Then, we fixed sample 1 in Methanol immediately, while the other two were submitted to FACS sorting with the gates set to include everything apart from very small debris and extremes in size and granularity. The samples were stored on ice for ~15 minutes during FACS setup. Sorting lasted 10 minutes per sample with no 4sU present in the FACS buffer (HBSS w/ 1% BSA). 10mm 4sU was added to the FACS buffer in the cooled receiving tube. Both samples were spun down and washed immediately after their sort before we resuspended the cells in ice cold HBSS (no 4sU added). Sample 2 was fixed in methanol directly afterwards, while sample 3 was left to incubate on ice for another 30 minutes in order to simulate waiting times when preparing multiple samples. Eventually sample 3 was also fixed using methanol. The cells were fixed at -20°C for at least 30 minutes. For IAA treatment the samples were brought back to RT and IAA dissolved in 20% HBSS / 80% MeOH (Th. Geyer #1437.2511) was added to a final concentration of 10 mM IAA. After converting for 16 hours, the cells were spun down at 1000 g and 4°C for 10 min, the supernatant was removed and quencing buffer added (DBPS, Gibco #14190144, 0.1% BSA, 1 U/µl RNaseOUT, Life Technologies #10777019, 100 mM DTT, Carl Roth #6908.1). We incubated the sample at RT for 5 min on order to quench the remaining IAA. Afterwards the cells were spun down again and resuspended in a small amount of resuspension buffer (DPBS containing 0.01% BSA, 0.5 U/ µl RNaseOUT and 1 mM DTT) and loaded onto the chromium controller using a Chromium Single Cell 3' v3.1 kit (10x Genomics #1000121). After successful encapsulation we proceeded with library preparation according to the manufacturer's instructions.