Nucleospin rna mini kit

Total RNA was extracted from each cell population using the NucleoSpin RNA Mini kit (MACHEREY-NAGEL). Then, secondary RNA purification was performed using the NEB Next Poly A m-RNA magnetic bead selection kit (E7490), and each cDNA library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770). Sequencing was performed using the Illumina Nextseq 500 platform with paired-end 75-bp reads. Sequenced reads were aligned to the mm10 reference genome using Bowtie2 (Langmead and Salzberg, 2012 (link)) with default parameters. Rsubread R package (Liao et al., 2019 (link)) was used to count the number of RNA-Seq reads. Screening of the differentially expressed genes, clustering analysis, and PCA analysis were performed using DESeq2 (Love et al., 2014 (link)) on the iDEP9.1 platform (Ge et al., 2018 (link)). Functional enrichment analysis was performed on Metascape (Mizukami et al., 2016 (link); Zhou et al., 2019 (link)).
The RNA-Seq datasets of FSCs or EpiSCs cultured in N2B27-based medium in the previous study (Kinoshita et al., 2021 (link)) were used for PCA analysis.

Total RNA was extracted from microglia cells according to the manufacturer’s instructions using the NucleoSpin® RNA Mini Kit (Macherey-Nagel, Dueren, Germany). RNA was quantified spectrophotometrically using a NanoDrop 2000 (Thermo Scientific) and then stored at −80°C. First-strand cDNA synthesis was performed with the RevertAid™ H Minus First strand cDNA Synthesis Kit (Thermo Scientific).

RT2-cancer cells were harvested by trypsin digestion and snap frozen in liquid nitrogen. RNA was prepared using the Nucleospin RNA Mini kit (Macherey-Nagel); cells were lysed using Tris(2-carboxyethyl)phosphine (TCEP)-containing RL1 buffer, followed by DNase digestion (Invitrogen). RNA quality was controlled by agarose gel electrophoresis and by OD600 measurements using a photometer (Eppendorf AG). Complementary DNA was prepared using the iScript cDNA synthesis kit (Bio-Rad Laboratories). Quantitative PCR was performed with SybrGreen using a LightCycler 480 (Roche). Gene expression was analysed using qbase software (Biogazelle) based on the delta-delta-CT-method and reference genes were evaluated using geNorm (feature of the q base software, Biogazelle). The following primers were used: SV40-Tag sense 5′-tcc act cca caa ttc tgc tct-3′, antisense 5′-ttg ctt ctt atg tta att tgg tac aga-3; Cdkn2a sense 5′-ttg ccc atc atc atc acct-3′, antisense 5′-ggg ttt tct tgg tga agt tcg-3′; Actb sense 5′-cta agg cca acc gtg aaa ag-3′, antisense 5′ acc aga ggc ata cag gga ca 3′; Eef1a1 sense 5′ aca cgt aga ttc cgg caa gt 3′, antisense 5′ agg agc cct ttc cca tctc 3′.

Total RNA of V. destructor samples was purified from 50 µl of homogenates of adult and immature stages using the NucleoSpin RNA-Mini kit (Macherey Nagel, Düren, Germany), following the manufacturer’s instructions. The 260:280 absorbance ratios (A260:A280) were measured using a BioTek Instruments analyzer (Winooski, VT, USA) analyzer. For the bee samples, total RNA was purified according to the manufacturer’s instructions from 140 µl of clarified pupa head homogenates (1 bee head per 500 µl PB) using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Viral particles were quantified in bee-pupae heads as previous studies highlighted that this is a clinical sign of overt DWV infection [36 (link), 55 (link), 61 (link)]. The extracted RNA was eluted from a spin column in 60 µl of DEPC-treated water or elution buffer (Qiagen). The RNA recovery rate from bee-head sample was not estimated because carrier RNA was used to enhance RNA purification efficiency. Next, 11 µl of pure undiluted RNA extract (about 159 ng of mite RNA) was transcribed into first-strand complementary DNA (cDNA) using the Superscript IV First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). The final volume of 20 µl of cDNA was kept at − 20 °C until the final quantification step was performed.

For HIF-1α qRT-PCR, PBMC and CBMC were stimulated for 48 h with anoxia or E. coli, harvested on ice and washed with ice-cold PBS. Cell pellets were frozen at −80 °C until further analysis. Total RNA was extracted using the NucleoSpin RNA Mini kit (Macherey Nagel, Düren, Germany) following the manufacturers’ instructions. RNA concentration and quality were measured in a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific).
For cDNA synthesis, 300 µg RNA were applied. After addition of random hexamer primers (Thermo Fisher Scientific), samples were heated to 65 °C for 5 min followed by cooling down to 4 °C. Then, a master mix (consisting of fivefold reaction buffer, ribolock RNase inhibitor, dNTPs and reverse transcriptase, all from Thermo Fisher Scientific) was added and the samples were heated again (25 °C for 5 min, 42 °C for 60 min, 70 °C for 5 min). The final cDNA-concentration was calculated to 50 ng/µl. At the end, samples were diluted with HPLC water in a 1:1 ratio. Real-time qRT-PCR reaction was performed using a LightCycler® 480 Instrument II (Hoffmann-La Roche AG, Basel, Switzerland). Prefabricated primers for HIF-1α (unique assay ID: qHsaCEP0050075), EGLN1 (unique assay ID: qHsaCEP0058182), and RPL37A (unique assay ID: qHsaCEP0052538) were purchased from Bio-Rad Laboratories.
RPL37A was used as house-keeping gene and data were evaluated via ΔΔCt-method.

Selected genes of interest were quantified using real-time reverse transcriptase quantitative polymerase chain reaction (qPCR). From cell culture, CD4⁺ T cells were harvested, and mRNA isolated using NucleoSpin RNA Mini Kit (MACHEREY-NAGEL, Cat# 740955.5) according to the instructions. The cDNA was transcribed using Promega M-MLV Reverse Transcriptase (Cat# M1701), RNasin Plus Ribonuclease Inhibitor (Cat# N2611), oligo(dT) 15 Primer (Cat# C1101), and PCR Nucleotide Mix (Cat# C1141). SYBR Green PCR Master Mix (Applied Biosystems, Cat# 4309155) was used for qPCR analysis, the primers used are detailed in Table S1.