title: GSM2526317: Control_3; Danio rerio; RNA-Seq
library_construction_protocol: Approximately 50 3 dpf larvae were collected in 2 ml eppendorf tubes, the E3 embryo medium removed, replaced by 10 mM DTT (Sigma) in E3 plus 0.2% tricaine and incubated for 1hr at RT. DTT preincubation is necessary to degrade the protective mucus layer around the larvae and enable larval tissue digestion. Larvae were washed twice in Ca2+ and Mg2+ containing PBS and subsequently incubated in 5 mg/ml collagenase I (Gibco) Ca2+/Mg2+ PBS for 3 hrs at 32C. Collagenase was removed by gently washing the larvae twice in DMEM containing 10% FBS. Using a 1000 ul pipette, the larvae were gently titurated and transferred into a 3 cm petri dish under a dissecting microscope equipped with epifluorescence. At this stage, the larvae are disintegrated into chunks of tissue with intact, bright GFP-positive proximal tubules floating free of adhering tissue. Pure proximal tubules were collected and placed in lysis buffer of the NucleoSpin RNA XS kit (Macherey-Nagel), and instantly frozen to -80C until further processing. RNA libraries were prepared using the TruSeq stranded total RNA kit for sequencing using standard Illumina protocols