Maxwell simplyrna kit

RNA was extracted using Maxwell simplyRNA kits and a Maxwell 16 Instrument (Promega). For the experiments that used miRNA mimics, the total RNA (including small RNAs) was extracted from the collected cells using miRNeasy Mini kits (Qiagen) in accordance with the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific). All cDNA used in mRNA and miRNA quantitative real-time PCR (qPCR) analyses was synthesized from extracted RNA by using the SuperScript VILO cDNA synthesis kit (Life Technologies) according to the manufacturer’s protocol. The mRNA and miRNA expression data were generated using Applied Biosystems TaqMan assays (20×) and Fast Advanced Master Mix (Life Technologies). Thermal cycling for qPCR was performed with an Applied Biosystems 7900HT Fast Real-Time PCR system (Life Technologies) in accordance with the TaqMan Fast protocol.

RNA extraction and DNAse treatment was performed using Maxwell simplyRNA kit (Promega) or RNeasy Mini kit (Qiagen), following the manufacturers’ instructions. In total, 200 ng of total RNA were reverse-transcribed with Superscript III (Invitrogen) following the manufacturer’s recommendations.
PCR reactions were carried out using GoTaq enzyme (Promega) with 1 μl of cDNA diluted 1:5. To quantify inclusion of alternatively spliced exons, capillary electrophoresis was performed using a Labchip GX Caliper workstation (Caliper, Perkin Elmer) at the CRG Protein Technologies Core Facility. The nanomolar content of each band was extracted with LabChip GX software and PSI values were calculated as the ratio between the inclusion amplicon and the sum of inclusion and skipping amplicons.
Real-time quantitative PCR (RT-qPCR) was performed on a ViiA7 Real Time PCR System (Applied Biosystems). Reactions in a total volume of 10 μl contained 2× SYBR Green Master Mix (Applied Biosystems), primers 400 nM and 1 μl of previously synthetized cDNA, diluted 1:5–20. The output Ct values were normalized by the expression of the housekeeping gene Gapdh (unless differently stated) and analyzed with ∆Ct/∆∆Ct method. All primers sequences are listed in Additional file 7: Table S6.

RNA was manually extracted from collected PAXgene Blood RNA tubes (Qiagen) with the PAXgene blood RNA kit (Qiagen) according to the manufacturer’s instructions or on an automated Tecan Freedom EVO 150 robotic platform with the Promega Maxwell SimplyRNA kit, using a modified protocol in a biosafety level 2 laboratory. Manually extracted RNA was stored at −80 °C, and later used for transcriptomic analysis. For RNA extracted by robotic platform an aliquot was immediately used for cDNA synthesis. For the Peruvian cohort, RNA samples were extracted from 10⁶ PBMCs using the RNeasy kit (Qiagen) according to manufacturers’ instructions, and blinded, frozen aliquots of RNA were shipped to the University of Cape Town.
For the venous versus capillary blood comparison, RNA was isolated with the PAXgene blood RNA kit (Qiagen) according to the manufacturer’s instructions with the following modifications: capillary blood samples were washed in 400 μL water (instead of 4 ml) and homogenised by pipetting to avoid loss of the small pellet; venous and capillary samples were eluted in 80 μL and 40 μL of PAXgene blood RNA kit elution buffer, respectively.

For RNA-seq of BJ cells depleted or not of SWINGN, total RNA was isolated and DNAse I-treated using Maxwell Simply RNA kit (Promega). ASO_LINC#3 was used to knockdown SWINGN expression. One microgram of quality-verified RNA was used for library preparation and sequenced on Illumina Nextseq 500 (75 bp single-end mode, 10 × 10⁶ reads/sample). Sequencing data were aligned to the genome assembly hg19 using STAR^{55 (link)} with default parameters. Differential expression analysis was carried out by using edgeR^{56 (link)} in R/Bioconductor and significant genes were selected applying the following filters: adjusted p-value < 0.01; |log₂FC| > 0.5.

Total RNA and cDNA were prepared using a Maxwell Simply RNA kit (Promega, Madison, WI, USA) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed with a Thermal Cycler Dice Real-Time System (TaKaRa Bio, Shiga, Japan) using SYBR Premix EX Taq (TaKaRa Bio). The relative levels of the mRNAs of interest were calculated using the 2^−ΔΔCt method. The mRNA level of Gapdh was used as the internal control. The primer sequences are listed in Supplementary Table S2.

Transcription of PunPgp-2 and PunPgp-9 was analysed by RT-PCR. Yeast were grown in induction media containing galactoses (SC-U medium but substituting the carbon source to 2% galactose and 1% raffinose) which induces activation of the GAL1 promotor and results in expression of the inserted gene. After 24 h of incubation at 30 °C and 250 rpm, total RNA was extracted using the Maxwell Simply RNA kit (Promega). cDNA was synthesized using the Maxima H- First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) from ~ 1 µg of RNA using Oligo(dT)₁₈Primers according to the manufacturer’s instructions. RT-PCR was performed using 0.02 U/µL Phusion Hot Start II polymerase (Thermo Fisher Scientific), 0.5 µM each primer, each dNTP at 200 μM, 1–2 ng/µL cDNA in a total volume of 25 µL 1 × HF buffer for two different primer sets (Primers in Supplementary Table S2). After a denaturation at 98 °C, 35 cycles of 98 °C for 10 s, a primer pair-specific annealing temperature for 15 s and 72 °C for 30 s were performed. Amplified PCR product were visualised after gel electrophoresis with GrGreen.