Ribo zero removal kit

An Epicenter Ribo-Zero™ Removal Kit (Epicenter, Madison, WI, USA) was used to remove rRNA, and rRNA-free residues were cleaned by ethanol precipitation. Then, sequences that passed the quality inspection were used for library construction and sequencing. The lncRNA and mRNA library was constructed using 3 μg of the total RNA and a NEBNext^® UltraTM Directional RNA Library Prep Kit (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions. The Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used for PE150 (paired-end 150 bp) sequencing. The miRNA library was built using a Small RNA Sample Prep Kit (Tiangen, Beijing, China). The total RNA was used as a template, and cDNA was synthesized with a special adapter. After PCR amplification, the products were purified by polyacrylamide gel electrophoresis (PAGE), and 140–160-bp sequences were recovered to construct an miRNA sequencing library. The above platforms were used for SE50 (single-end 50 bp, SE50) sequencing. All the sequencing were outsourced to Wuhan Frasergen Gene Information Co., Ltd. (Wuhan, China).

Samples from five layers (0 to 2 cm, 6 to 8 cm, 12 to 14 cm, 20 to 22 cm, and 28 to 30 cm) were selected for metatranscriptomic sequencing. Total RNA was isolated from 10 to 20 g of sediment using an RNeasy PowerSoil total RNA kit (Qiagen). Genomic DNA was removed using a Turbo DNA-free kit (Ambion), and rRNA was removed using a Ribo-Zero removal kit (Illumina). The purified RNA was sequenced at Novogene Bioinformatics Technology Co., Ltd. (Tianjing, China), on an Illumina HiSeq 2000 using the PE150 strategy (paired-end reads of 2 × 150 bp). Approximately 220 gigabase pairs (Gbp) of raw sequence data were obtained for each sample.
Raw reads from metatranscriptomic sequencing were quality trimmed and filtered using Sickle (https://github.com/najoshi/sickle). SortMeRNA v4.3.4 (93 (link)) was used to filter rRNA and tRNA fragments from metatranscriptomic data. The paired-end reads from metatranscriptomic sequencing were mapped onto the predicted gene sequences of strain FT118^T using BWA mem (94 (link)) with the default setting. The coverage information was extracted using SAMtools v1.3.1 (95 (link)) and bedtools v2.30.0 (96 (link)). The transcript per million (TPM) values were calculated to determine gene expression activity in metatranscriptomes.