Rneasy protocol

RNA was isolated in triplicate from independent fly crosses for each experiment and was prepared from purified cells according to the Qiagen RNeasy protocol. A total of 50 ng of total RNA sample was used as starting material to make cRNA probes followed by NimbleGen probe labeling. Double-stranded cDNA synthesis was carried out using Superscript III (Invitrogen 18080–051) followed by cRNA amplification using an Epicenter TargetAmp 1-Round aRNA amplification kit (TAU1R5124). Double-stranded cDNA was synthesized again and further labeled using a NimbleGen One-Color DNA labeling kit (05-223-555) and then hybridized to a D. melanogaster Gene Expression 4 × 72K Array (A4509001-00-01) according to the NimbleGen expression array protocol. Arrays were scanned using a GenePix 4000 microarray scanner and data was extracted using NimbleScan v2.4 software (Roche NimbleGen Inc, Madison, WI, USA). Microarray data was processed in NimbleScan using default settings. Data was further uploaded into the ArrayStar software version 3 (DNASTAR, Inc, Madison, WI, USA) for normalization and statistical analysis. A moderated t-test was performed with false discovery rate (FDR; Benjamini–Hochberg), and multiple testing corrections were applied on data (p<0.05) with a 2-fold change or greater.

Total RNA was extracted using the RNeasy protocol (Qiagen). RNA concentration was determined by
measuring absorbance at 260 nm. Equal RNA levels were used to generate complementary DNA
using the high-capacity cDNA reverse transcription protocol (Applied Biosystems). Quantitative PCR
was performed using reaction mixtures of 8.4 ng total RNA, 100 nM primer, and SYBR
Green reagent (Applied Biosystems).

RNA isolation was performed using two biological samples per time point and the Qiagen RNeasy protocol. Four micrograms of RNA was employed to generate the RNA-seq libraries. In short, total RNA was subjected to rRNA depletion using the Ribo-ZeroTM Gold kit (Epicentre) according to the manufacturer's instructions. rRNA-depleted samples were employed for first- and second-strand cDNA synthesis and using dUTP for strand-specificity. RNA-seq libraries were prepared using KAPA Hyperprep kit (Roche 07962363001) according to the manufacturer's instructions and the final libraries were sequenced on a HiSeq-2000 Illumina sequencer.

RNA isolation was performed according RNeasy protocol (QIAGEN, Germany), followed by reverse transcription using the Qscript cDNA synthesis kit (Quanta BIO, U.S.). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR or QPCR) analysis was performed using SYBR green mix (Applied Biosystems, U.S.) to describe transcript levels of different genes. The details of the sequences and thermal cycling conditions were according to the standard protocol. Data were acquired and analysed with IQ5 software (Bio-Rad, U.S.).

DNA and RNA were isolated from alternating sections (10 µm) of fresh frozen tumor samples. A standard proteinase K digestion, followed by a chloroform extraction was used to obtain DNA of high molecular weight. Isolation of RNA was performed using Trizol (Invitrogen, Carlsbad, CA, USA) and a subsequent automated RNeasy protocol (QIAgen, Hilden, Germany). The quality of the RNA was assessed on a BioAnalyzer (Agilent, Santa Clara, CA, USA). Only samples with a RNA Integrity Number (RIN) score higher than 7.5 were included. Human primary fetal osteoblasts were cultured to a confluency of 90% and RNA was isolated to act as a reference in the microarray expression analyses.

We used the RNeasy protocol for isolation of total RNA from CD34⁺ cells according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). Concentration and integrity of total RNA were assessed using NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, Delaware, USA) and Agilent 2100 Bioanalyzer Software (Agilent Technologies, Waldbronn, Germany) comparing the ratio of 28S and 18S RNA peaks to ensure that there is minimal degradation of the RNA sample.