Split rna extraction kit

Msm cultures were grown in 7H9 at 37 °C to the stated OD₆₀₀ before application of the stressor followed by continued incubation for the indicated time. Cells were harvested by centrifugation and then lysed in 2 ml screw cap tubes containing 500–700 mg 0.15 mm zirconium oxide beads using a Minilys personal homogeniser. The lysate was cleared by centrifugation at 20,000 × g for 10 minutes and the total RNA purified using a SPLIT RNA Extraction kit (Lexogen) and following the manufacturer’s instructions. Removal of DNA was achieved using the Turbo DNase kit (ThermoFisher Scientific) and the RNA concentration measured using the NanoDrop™ One spectrophotometer. 100 ng RNA was reverse transcribed to complementary DNA (cDNA) using iScript Select cDNA Synthesis kit (BioRad). Primers for qPCR were designed using Primer3 software^{59 (link)} and are listed in Supplementary Table 1b. qPCR was performed using the Kapa Sybr Fast qPCR kit (Roche) in a BioRad CFX96 thermocycler with an initial 5 minutes at 95 °C step followed by 40 cycles of 95 °C for 10 seconds, 60 °C for 10 seconds, 72 °C for 12 seconds. Data were analysed using BioRad CFX Maestro Software. Relative gene expression was determined using the 2^-ΔΔCt method and with rpoB or sigA as the reference gene.

Total RNA was purified from approximately 50 mg of fresh mycelium using SPLIT RNA Extraction Kit (Lexogen, Austria) and treated with DNase I (ThermoFisher Scientific). RNA quantity and quality were checked using respectively a Qubit^® 2.0 Fluorometer (Invitrogen) and Tape Station 4200 (Agilent) resulting in a RNA integrity number (RIN) of 10.
Approximately 1 μg of total RNA eluted in RNase-free water was sent to Fasteris SA (Plan-les-Ouates, Switzerland) for RNA library construction and deep sequencing. The library preparation was performed using the Illumina TruSeq^® stranded RNA Sample Preparation Kit (Illumina, San Diego, CA, United States). The library was sequenced in pair-end (2x 75 nt) runs on an Illumina NextSeq 500 machine. Prior to the library preparation, Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) was used successfully. An “in-lane” PhiX control spike was included in each lane of the flow-cell. 93.94% of the reads had a quality value (Q30) ≥ 30, i.e., less than 1 error in 1000 bases. The raw data was deposited in the Sequence Read Archive (SRA) with the BioProject ID PRJNA619952 and BioSample ID SAMN14856044.

To analyze adipogenesis-related gene expression in vitro and in vivo models, the total RNA was isolated from the samples using the SPLIT RNA Extraction Kit (Lexogen, Vienna, Austria) based on the product manual. Extracted total RNA was further purified with Ambion™ RNase Inhibitor (Applied Biosystems) and TURBO DNA-free™ Kit (Applied Biosystems), and then saved for further cDNAs synthesis. cDNAs were synthesized from 1 mg total RNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). cDNAs were then analyzed by qPCR using a Power SYBR Green PCR Master mix (Applied Biosystems), which is performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) for qPCR analyses. Specific primer sequences were adopted from Breasson et al. (2017) (link). Relative quantification of gene expressions was performed using the comparative Ct method and normalized by an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results were expressed as fold difference relative to a relevant control sample.

Before RNA extraction, samples were additionally centrifuged at 3,000 g at 4°C for 15 min and the obtained supernatant was used for RNA extraction. Small RNAs were extracted using SPLIT RNA Extraction Kit (Lexogen) according to manufacturer's recommendations.

Large molecular weight RNA was differentially isolated from the small molecular weight RNA fraction using the SPLIT RNA extraction kit (Lexogen, Vienna, Austria) as per the manufacturer’s instructions. RNA quality was evaluated using the Agilent RNA 6000 Nano Kit on a Bioanalyser 2100 (Agilent Technologies, Santa Clara, CA, USA). Only RNA samples with an integrity number ≥7.0 were kept for RNA-seq library construction. Each sample was quantified using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and, prior to constructing RNA-seq libraries, 1.5 µg of RNA was enriched in poly-A RNA using magnetic beads with poly-T oligonucleotides. Enriched poly-A RNA was used to construct libraries using the Illumina TrueSeq RNA sample prep kit v2 (llumina, San Diego, Ca, USA) as per the manufacturer’s instructions except that the RNA fragmentation step was performed during six minutes. Replicate libraries were indexed with a unique barcode identifier and then quantified and mixed to form a normalized 12-plex paired-end sequencing library. A single lane (50-nt paired-end reads) of an Illumina Hi-Sequation 2000 instrument (Illumina Inc, San Diego, CA, USA) was used to sequence the entire library at McGill University-Genome Quebec Innovation Centre (Montréal, Canada).