Dreamtaq kit

DNA was isolated from two single male and female flies using the Qiagen DNeasy Blood/Tissue kit. PCR primers were designed using the Primer3 software based on assembled putative Y-linked transcripts. PCR amplification was performed with the ThermoFischer Scientific DreamTaq kit, with annealing temperatures ranging from 55 to 60 °C.

RNA of tissue samples was purchased from Ambion (Life Technologies). RNA of cell lines was extracted using TriPure Isolation Reagent (Roche Diagnostics GmbH). Reverse transcription was performed on 2 μg of total RNA using M-MLV Reverse transcriptase and random hexamers (Invitrogen). For PCR reactions, we used the DreamTaq Kit (Thermo Fisher Scientific), incorporating 1/40 of the reverse transcription mixture in a final reaction volume of 20 μl. PCR reactions were visualized after electrophoresis in an ethidium bromide-stained agarose gel. For qPCR reactions, we used KAPA SYBR FAST (Sigma-Aldrich), incorporating 1/40 of the reverse transcription mixture in a final reaction volume of 10 μl. All reactions were carried out according to the manufacturer’s instructions. All primers are listed in the supplementary table S2.

RNA of normal tissue samples (lung, testis, esophagus and colon) was purchased from Ambion (Life Technologies). RNA of normal and tumor cell lines was extracted using TriPure isolation reagent (Roche, Basel, Switzerland). Reverse transcription was performed using MML-V reverse transcriptase kit (Invitrogen), random hexamers (Invitrogen), Ribolock RNAse inhibitor (Invitrogen, 20U) and 2 µg of total RNA per reaction in a final volume of 20 µL. PCR analyses were carried out using DreamTaq kit (ThermoFischer Scientific, Waltham, MA, USA) with 1/40 of the reverse transcription solution engaged per reaction in a total volume of 20 µL. qPCR analyses were performed using KAPA SYBR FAST kit (Sigma-Aldrich) with 1/40 of the reverse transcription solution engaged per reaction in a final volume of 10 µL. All reactions were carried out according to the manufacturer’s instructions. Primer sequences and experimental conditions are listed in Table S7. Different positive control cDNAs were used depending on the gene: testis (NAP1L1, NAA11, MAGEA1, ACTB), colon (EPS8L3, VIL1), esophagus (GJB5, SERPINB5, PGLYRP3).