Nebnext rna library preparation kit

The procedures for sample collection, DNA and RNA library preparation for most datasets (DNA shotgun and mate-pair libraries and an inflorescence transcriptome library) and sequencing were described in the reports of Logacheva et al. (2016) (link) and Schelkunov, Penin & Logacheva (2018) (link). In addition to the data generated in previous studies, we used four new datasets that represent the transcriptomes of the anthers and ovules of H. monotropa. The samples were collected in the same location as previous samples but in 2018. H. monotropa is not an endangered or threatened plant species; thus, no specific permissions were required for its collection. RNA was extracted using an RNEasy kit (Qiagen, Hilden, Germany) with the addition of the Plant RNA Isolation Aid reagent (Thermo Fisher, Waltham, MA, USA) to the lysis buffer. The removal of ribosomal RNA was performed using a Ribo-Zero plant leaf kit (Illumina, San Diego, CA, USA), and further sample preparation was performed using a NEBNext RNA library preparation kit (New England Biolabs, Ipswich, MA, USA). The libraries were sequenced on the HiSeq 4000 platform (Illumina, San Diego, CA, USA) in 150-nt paired-end mode at the Skoltech Genomics Core Facility. The reads were deposited in the NCBI Sequence Read Archive under BioProject PRJNA573526.

The whole‐transcriptome library was prepared using a Ribo‐Zero Magnetic Gold Kit (Illumina) and a NEBNext RNA Library Preparation Kit (New England Biolabs). Extracted RNA from lung tissues was quality‐controlled and quantified on a BioAnalyzer 2100 Systerm (Kapa Biosystems). The library sequencing was performed on a HiSeq 2000 Instrument (Illumina). According to the instructions, 1 μg of total RNA was used for RNA library preparation. The fragments less than 10 nt or over 34 nt were discarded. The pure reads were compared to the miRNA precursors in the miRBase 22.1 to validate the outcomes.

The murine lung tissues were pre-hybridized with DNA. The whole transcriptome libraries were prepared using a Ribo-Zero Magnetic Gold kit (MRZE126, Zhongbeilinge Co., Ltd., Beijing, China) and a NEBNext RNA library preparation kit (E7775, New England Biolabs, Beverly, MA, USA). A BioAnalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA) was used for quality control and quantification. The obtained libraries were sequenced on a HiSeq2000 kit (Illumina Inc., San Diego, CA, USA) to analyze differentially expressed mRNAs. The volcano map for differentially expressed genes was produced using an R package (NIH, Bethesda, MA, USA). All procedures were conducted based on the manufacturer’s instructions [25 (link)].