Superscript double stranded cdna synthesis kit

For each individual, a supragingival plaque sample was collected from all surfaces of all teeth by using a sterilized toothpick. Professional tooth cleaning was stopped for 1 month, and patients were instructed to stop their own plaque control at least 1 h before the sample collection. The collected plaque was placed into PM1 buffer in the PowerMicrobiome RNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) in a sterile tube, and was stored at − 80 °C. The RNA extraction, cDNA synthesis, library preparation, and Illumina sequencing were conducted, as described previously [18 (link)] with the following modifications. The SuperScript® Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and 6-mer random primer were used instead of the SMARTer Ultra Low RNA kit (Clontech, Mountain View, CA, USA). MiSeq (Illumina, Inc., San Diego, CA, USA) reads were generated as 300-bp paired-end.

Equal amounts of high-quality RNA samples were used to synthesize cDNA libraries. Briefly, mRNAs were purified from total RNAs and used as templates to synthesize the first-strand and the second-strand of cDNAs, according to the protocol of Super Script Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, MA, USA). cDNAs were cut into short fragments following the TruSeq RNA sample preparation guide. After end repair and the addition of poly (A), the short fragments were ligated with sequencing adapters and enriched by PCR amplification to construct the cDNA library templates. Finally, transcriptome sequencing was performed using the Illumina HiSeq 2000 platform, which generated about 100-bp paired-end (PE) raw reads.

Double-stranded cDNA was prepared from 2 µg of total RNA extracted from stool samples using Superscript^® double stranded cDNA synthesis kit (ThermoFisher Scientific) according to manufacturer’s instructions, except that the first strand cDNA synthesis was primed using random hexamers (Promega). Double stranded cDNA (200 ng) was sheared using Covaris S2 sonicator in 52.5 μl volume with the following parameters: 10% duty cycle, intensity 4 and 200 cycles per burst for 70 s. Subsequently, 17 µl of sheared cDNA (65 ng) was end-repaired, A-tailed, adapter-ligated, Ampure XP beads-purified and libraries were PCR-enriched as described above for metagenome library preparation. PCR-enriched libraries were cleaned up and size-selected to remove unused dNTPs, primers and short RNA fragments, using a 0.65 × ratio of Ampure XP beads (Beckman Coulter).

Mycelial samples from the one litre bioreactors were collected by vacuum filtration on Whatman GF/A filter paper and then rinsed in 0.9% NaCl and frozen immediately in liquid nitrogen. RNA was isolated from 50 mg of the mycelia with RNeasy Plant RNA isolation kit (Qiagen GmbH. Hilden, Germany). RNA quality was assessed for purity and quantity using an Agilent RNA 6000 analyser (Agilent Technologies Inc. Santa Clara, CA, United States of America) The purified RNA was then converted into cDNA using an Invitrogen Superscript Double-Stranded cDNA Synthesis Kit (Thermo) prior to labelling with Cy3 One-Colour fluorescent dye (Roche NimbleGen Inc. Basel, Switzerland) according to the manufacturer’s instructions. The labelled cDNA was hybridised on an automated HS12 system (Roche) to custom-made four plex microarray slides (Roche) designed from the T. reesei genome version 2.0 [23 (link),24 (link)]. Analysis was performed in an MS200 microarray scanner (Roche) to identify signal probe intensity of the six 50-74mer probe replicates designed from the known and predicted genes in T. reesei.

Each replicate V. fischeri WT and ∆hfq culture was thawed on ice and pelleted for RNA extraction. Total RNA was extracted in triplicate for each treatment using PowerSoil^® Total RNA Isolation Kit (Qiagen, Germantown, MD) according to manufacturer’s protocol and was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA) to remove potential contaminating DNA. The RiboMinus rRNA removal kit (Thermo Fisher Scientific, Waltham, MA) was used to deplete large rRNAs and samples were processed with the Zymo RNA Clean & Concentrator kit (Zymo Research, Irvine, CA). The remaining mRNA was pooled between replicates, the concentration was determined by Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA) and quality was evaluated with a 2100 Bioanalyzer generating RIN factor > 9 (Agilent Technologies, Santa Clara, CA). High-quality RNA was converted to cDNA using a modified SuperScript Double Stranded cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA). A total of three replicate cDNA libraries were generated for each treatment (note: only two libraries were generated for ∆hfq gravity controls) and sequenced using the Illumina NextSeq500 platform (2 × 150 bp paired-end reads; Illumina, San Diego, CA).

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according the manufacturer’s instructions. RNA concentration was determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was determined by agarose gel electrophoresis. Poly(A) mRNA was isolated from the total RNA using Oligo (dT) and cleaved into short fragments using a Covaris S220 Focused-Ultra sonicator (Covaris, California, USA) and then used as templates for the synthesis of first- and second-strand cDNA according to the protocol of the Super Script Double-Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA). The cDNA was purified using a QIAquick PCR Purification Kit (QIAgen, Düsseldorf, Germany). After end repair, poly(A) addition and sequencing adapter ligation, the optional 300–400 bp fragments were selected by agarose gel electrophoresis, and enriched by PCR amplification to construct the cDNA library, followed by sequencing and generation of 150 bp paired-end reads using the Illumina HiSeq Ten platform. Three independent biological replicates were performed for stage Ⅱ and Ⅳ based on each tissue.