Tri reagent

Individual eyed eggs were homogenised in 1 mL Tri Reagent (Sigma-Aldrich^®) using a Mini-Beadbeater-24 (BioSpec Products Inc.) and RNA was extracted following the manufacturer’s standard Tri Reagent protocol. RNA quantity and quality of individual embryos were assessed by spectrophotometry (NanoDrop ND-1000) and agarose gel electrophoresis, respectively. Each extraction yielded about 40 to 50 µg RNA. For each hybridisation sample (biological replicate), equal amounts of total RNA from eight individuals (four per family × two families) were pooled per reciprocal cross type (WW, DD, DW or WD) and then re-quantified and quality-assessed as described above (Fig. 1).Fig. 1

Schematic representation of the experimental design

Lungs were homogenized in TRI Reagent with 1.0 mm diameter zirconia beads (Biospec Products) for 30 seconds using bead beater disruption (Minibeadbeater-16, BioSpec Products). RNA was extracted as described in the manufacturer’s protocol (Direct-zol RNA miniprep kit, Zymo Research). First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) at 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, followed by a cooling step at 4°C. qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems) with the following thermocycling program: [95°C for 5 min, 95°C for 15 sec, 55°C for 1 min] x 40 times, followed by 65°C to 95°C (0.5°C increment) for 5 sec (C1000 Touch Thermal Cycler, Bio Rad). Hprt1 was used as the housekeeping gene. During RNA isolate, RNA was treated with DNase using the Direct-zol RNA miniprep kit from Zymo Research. RNA (without reverse transcriptase treatment) was tested as a genomic contamination control. 1 ug of total RNA was used for the first strand cDNA template synthesis to generate 20 μl of cDNA. cDNA was dilution by a factor of 4 and 1 μl was used in a 10 μl RT-PCR reaction. Primer sequences are provided in S1 Table.

Coverslips containing cells were moved to an empty well in a new plate. One millilitre of TriReagent (TR118; Molecular Research Inc.) was added and, after pipetting several times, the mixture was moved to a 2 ml BioSpec tube (5225; Bio Spec Products Inc.) and stored in a ‐80°C freezer. At the end of the experiment, all samples were thawed, and RNA purified with added glycogen as previously described (Bechshøft et al. 2019 (link)).
For the tissue samples, 100 sections (10 μm each) from the frozen biopsies were transferred to the 2 ml BioSpec tubes and dissolved in 1 ml TriReagent by shaking with five steel beads (2.3 mm, BioSpec) for 15 s in a FastPrep homogenizer (MP Biomedicals). The RNA was purified as for the cell culture, except no glycogen was added.