Truseq stranded mrna lt sample prep kit

Biomass sampling was performed when switching incubation pressure (Fig. 1C). Total microbial genomic DNA was extracted and purified using the modified SDS-based method described by Natarajan et al.^{43 (link)}, and stored at −20 °C before further assessment. The quantity and quality of extracted DNA were measured using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed to construct metagenome shotgun sequencing libraries with insert sizes of 350 bp following the standard Illumina TruSeq DNA Sample Preparation Guide. Each library was sequenced by Illumina NovaSeq 6000 platform (Illumina, USA) with PE150 strategy at Shanghai Personal Biotechnology (Shanghai, China). The extraction of RNA from sediment samples was carried out using the RNeasy® PowerSoil® Total RNA Kit (Cat. No. 12866-25, Qiagen, Germany) according to the manufacturer’s instructions, then quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). To ensure DNA removal, the RNA extracts were treated with TURBO DNase (Cat. No. AM2238, Invitrogen, Waltham, MA, USA) as directed by the manufacturer. The purified RNA was converted to cDNA, then the metatranscriptomic library was constructed by using Illumina TruSeq Stranded mRNA LT Sample Prep Kit, and subsequent sequencing as described above.

To assist the assessment of genome assembly and genome annotation, we sequenced transcriptome data from the first‐ and second‐stage larvae and adult flies of W. magnifica with three replicates for each sample. Total RNA was extracted from each sample. RNA quality was examined by agarose gel electrophoresis and Agilent 2100 Bioanalyser (Agilent Technologies). Library preparation followed the instructions of TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). Briefly, mRNA was enriched by binding to poly‐A on mRNA with Beads containing oligo‐dT, and the enriched mRNA was interrupted to a 200–300 bp fragment. Then, the fragmented RNA was used as a template for reverse transcription to the first‐strand complementary DNA (cDNA) synthesis, followed by the synthesis of second‐strand cDNA, which uses the first‐strand cDNA as a template. After that, synthetic double‐stranded cDNA was end‐repaired, poly (A) added, and ligated to Illumina sequencing adapters. The ligation products were first purified by removing the free adaptor and the fragment without the attached adaptor, and next amplified by PCR using specific primers. Finally, the prepared libraries were sequenced on an Illumina NovaSeq platform. To obtain high‐quality clean reads, the raw paired‐end reads were trimmed by removing adapter sequences and low‐quality reads using bbmap (https://sourceforge.net/projects/bbmap/).

Total RNA was extracted using TRIzol reagent (Invitrogen) and extracted following the manufacturer’s protocol. For RNA sequencing, the libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA). And were sequenced on the Illumina sequencing platform (HiSeqTM 2500). For Pacbio sequencing, cDNA was synthesized using a SMARTer PCR cDNA Synthesis Kit (Clontech) and SMRT bell libraries were generated using a SMRTbell Template prep kit 1.0 (Pacific).

The libraries from the mouse total RNA were prepared using the TruSeq^® Stranded mRNA LT Sample Prep Kit (Illumina, Inc., Rev.E, October 2013) according to the manufacturer’s protocol. Briefly, 0.25 µg of total RNA was used for poly-A-based mRNA enrichment with oligo-dT magnetic beads. The mRNA was fragmented (resulting RNA fragment size was 80–250 nt, with the major peak at 130 nt) and the first strand cDNA synthesis was done by random hexamers and reverse transcriptase. The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, this allowed to achieve the strand specificity. The blunt-ended double stranded cDNA was 3′ adenylated and Illumina platform compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. Each library was sequenced on HiSeq4000 (Illumina) in a fraction of a HiSeq 4000 PE Cluster kit sequencing flow cell lane, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 2.7.7) and followed by generation of FASTQ sequence files.

Total RNA extractions and quality checks were performed, as described previously (annotation section). RNA samples were shipped to Novogene (Beijing, China) for sequencing. At arrival, samples were tested before library preparation and all passed the QC performed for quantitation (NanoDrop, ThermoFisher), degradation (agarose gel), and integrity (Agilent 2100, Bioanalyzer systems). Library preparation was performed with a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) and included rRNA depletion using the Ribo-Zero kit (Illumina). All samples passed the library QC performed for concentration (Qubit 2.0, ThermoFisher), insert size (Agilent 2100), and library effective concentration (qPCR). An Illumina HiSeq 2500 instrument was used for sequencing to obtain 150 bp paired-end reads.

Comparative transcriptomics of cells grown on CO and H₂/CO₂ was performed to investigate gene expression profiles based on three biological replicates. Cell pellets from cultures in the bioreactor were collected by centrifugation at 10000 × g under −4°C for 10 min at exponential phase and frozen in liquid nitrogen immediately and stored at −80°C. The RNA isolation and high-through RNA sequencing (RNA-Seq) were accomplished by Allwegenetech Corp (Beijing, China). Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Santa Clara, CA, United States) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bio-analyzer (Agilent Technologies, Santa Clara, CA, United States). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. Then these libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500) and 150 bp/125bp paired-end reads were generated. Based on reads per kilobase of transcript per million mapped reads (RPKM) normalization, the genes expression profiles were analyzed. The processed RNA-Seq data were submitted to the ArrayExpress database¹ under the accession number E-MEAB-8260.