Rneasy blood and tissue kit

Quantitative PCR was performed by standard procedures. Briefly, total RNA from 5*10⁶ primary CLL cells was isolated using the RNeasy Blood and tissue kit (Qiagen, Hilden, Germany). Subsequently, 500 ng of RNA were transcribed to cDNA using oligo dT primers (Superscript III, Thermo Fisher Scientific). Q-PCR was performed using specific primers for CD38, E2A and NF-kB. ERCC6 was used as loading control.
Primers used were: CD38 forward: 5´-CTGAGGATTCATCTTGCACATCTG-3´, CD38 reverse: 5´-GGCTTCCGTCTCTGGCATTG-3´, NFKB1 forward: 5´-GAAGCTGAAGTGCATCCAAAGG-3, NFKB1 reverse: 5´-GCCAGTGTTGTGATTGCTAGATAC-3´, E2A forward: 5´-TCCCTTCTCGGTGGCTTCC-3´, E2A reverse: 5´-CGCACAGTTCCAGAGGCTATG-3´. A reference gene assay was used for ERCC6 (Primerdesign Ltd., Southampton, UK).

We confirmed the efficacy of the IRS knockdown with quantitative real-time PCR. Abdominal fat bodies were dissected from seven-day old bees, flash frozen in liquid nitrogen, and stored at −80 °C until processing. We extracted RNA using TRIzol (GIBCO-BRL, San Diego, CA, USA)/chloroform extraction paired with RNeasy Blood and Tissue kit and an additional DNase treatment (Qiagen) as described previously [44 (link)]. RNA was quantified using two-step qRT-PCR, and analysed using the ΔΔCT method relative to expression of β-actin (GenBank: XM_623378) [50 (link)]. β-actin is stably expressed across several tissue types in adult honeybees and has been shown to be a reliable reference gene [51 (link),52 (link)]. Samples were run in triplicate along with a negative control (a reaction lacking reverse transcriptase) to ensure reliability and the absence of DNA contamination. Knockdown efficiency was established by comparing relative expression of irs in the IRS and GFP treatment groups from both strains. This allowed us to determine the effect of reducing irs expression via targeted dsRNA versus the procedure of injecting dsRNA itself as described before [11 (link),53 (link),54 (link)].

RNA from the proximal colonic section was isolated using a QIAGEN RNeasy Blood and Tissue kit according to the manufacturer’s instructions. The integrity and purity of RNA were verified by agarose gel electrophoresis and spectrophotometry using a NanoDrop ND-1000. RNA was reverse transcribed by SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative PCR (qPCR) was carried out with EvaGreen intercalating dye (Syntol, Moscow, Russia) on the CFX96 Touch™ Real-Time PCR Detection System (BioRad, Hercules, CA, USA). PCR conditions included a preheating step at 95 °C for 3 min and 40 cycles at 95 °C for 15 s, 57 °C for 15 s, and 72 °C for 15 s coupled with fluorescence measurement. The amplification specificity was confirmed by melt curves analysis. Each sample was run in triplicate, and a non-template control was added to each run. The choice of reference genes TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) was based on the results of a stability study of the reference genes in a mouse model of DSS-induced colitis [33 (link)]. The expression levels of the target genes were determined by the ΔΔCt method with the calculated primer efficiencies and normalized on the geometric mean of selected reference genes. Primer sequences are listed in Supplementary Table S2.

Total RNA was isolated from four human colon tissue samples using an RNeasy Blood and Tissue kit (Qiagen, Carlsbad, CA, USA). To construct the sequencing library for HiSeq 4000, we followed the TruSeq Stranded mRNA Sample Preparation Guide, Part #15031047 Rev. E. Approximately 2 μg of total RNA was used for library construction with the Illumina TruSeq Stranded mRNA Library Prep Kit (San Diego, CA, USA). Next, paired-end sequencing was performed using the Illumina HiSeq4000 sequencing instrument, according to the manufacturer’s instructions, yielding 101-bp paired-end reads. To construct the library for the MGISEQ-2000, approximately 1 μg of total RNA was used for library construction using the MGIEasy RNA Directional Library Prep Kit (MGI). Next, paired-end sequencing was performed using the MGISEQ-2000 sequencing instrument, according to the manufacturer’s instructions, yielding 100-bp paired-end reads. The RNA-seq data of HiSeq 4000 were generated in 2013, while the MGISEQ-2000 data were generated in 2019. Thus, although we used RNA from the same samples, the sequencing was not performed at the same time.

T. brassicae wasps from the S301 line were collected for RNAseq for evidence-based annotation. Hundreds of adult individuals (male and female) were collected and stored at -80°. For RNA extraction, samples were frozen in liquid nitrogen in a single 1.5 mL safelock tube with approximately six 1-mm glass beads and shaken for 30 s in a Silamat S6 shaker (Ivoclar Vivadent). The RNeasy Blood and Tissue Kit (Qiagen) was used according to manufacturer’s instructions, and final column elution was achieved using 60 µL sterilized water. The sample was measured for quality and RNA quantity using an Invitrogen Qubit 2.0 fluorometer and the RNA BR Assay Kit (Thermo Fisher Scientific). The RNA sample was then processed by Novogene Bioinformatics Technology Co., Ltd., (Beijing, China) using poly(A) selection followed by cDNA synthesis with random hexamers and library construction with an insert size of 300 bp. Paired-end sequencing was performed on an Illumina HiSeq 4000 according to manufacturer’s instruction. Quality filtering was applied to remove adapters, reads with more than 10% undetermined bases, and reads of low quality for more than 50% of the total bases (Qscore less than or equal to 5).