Zr duet kit

All dissections were performed between 1–4 pm to limit gene expression differences due to circadian rhythm genes. To isolate the Malpighian tubules, the penultimate abdominal segment of a cold-anesthetized female was gently pulled with a micro-tweezer until the gut, Malpighian tubules, and ovaries were isolated from the carcass. Following removal of the ovaries and foregut, the Malpighian tubules (and some hindgut) were then immediately transferred into lysis buffer (Zymo Research, Irvine, USA) and homogenized with a micropestle (Additional file 1: Table S1). Total RNA (and DNA in parallel) was then extracted from individual females or pools of 8–12 Malpighian tubules per biological replicate using the ZR-Duet kit (Zymo Research) with an on-column DNAse treatment step.

Low‐pass whole‐genome sequencing (WGS) was conducted as described in Yuan et al (2018). Briefly, we homogenized tissue with a Dounce and extracted DNA and RNA with a ZR‐Duet Kit (Zymo) according to the manufacturer's instructions. For the normal control, DNA and RNA were extracted using the same kit from peripheral blood mononuclear cells. WGS libraries were constructed by in vitro transposition using the Illumina Nextera XT kit and sequenced on an Illumina NextSeq 500 with 2 × 75 base paired‐end reads to a depth of ~ 1×. Reads were aligned to the hg19 build of the human genome using bwa‐mem, and the coverage for each chromosome was quantified using bedtools after collapsing PCR duplicates with samtools. To generate the bulk WGS heatmap in Appendix Fig S3E, we divided the normalized coverage of each chromosome in the tumor sample by that of the normal sample, normalized the resulting ratio by the median ratio across all chromosomes, and multiplied by two to estimate average copy number of each chromosome in the tumor sample. Note that we do not have consent to share the raw WGS data from these patients.

For each tumor, a 2–3-mm³ piece was used for DNA extraction. Each section was re-suspended in 400 μL of DNA/RNA Lysis Buffer (Zymo) and homogenized with a Dounce homogenizer if necessary. DNA and RNA were then extracted for the tissue using the ZR-Duet Kit (Zymo) according to the manufacturer’s instructions. DNA was quantified using the Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific). Libraries for low-pass WGS were constructed using by in vitro transposition the Nextera XT kit (Illumina). DNA inputs for each sample were normalized to 1 ng and library preparation was performed according to the manufacturer’s instructions, using unique i7 indices for each sample. Libraries from all eight tumors were pooled at equimolar concentrations, denatured, diluted, and sequenced on an Illumina NextSeq 500 using a 150-cycle High Output Kit (Illumina, 2 × 75 bp).