Nebnext multiplex small rna library prep set

Small RNA sequencing data from three biological replicates of T. castaneum adults used as controls and adults subjected to 24 h heat stress at 40 °C and a recovery period of 1 h were prepared using the NEBNext Multiplex Small RNA Library Prep Set. Sequencing was performed at GeneCore (EMBL, Heidelberg, Germany) by Illumina HiSeq2500 producing 50 bp long single-end reads and the sequencing data are deposited in the NCBI/bioproject database under accession number PRJNA679427 (https://www.ncbi.nlm.nih.gov/bioproject/679427). Small RNA sequencing data from oocytes and embryos at 0–5, 8–16, 16–20, 20–24, 24–34, 34–48 and 48–144 h were previously generated [25 (link)] and deposited in the GEO database, GSE63770 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63770). Sequencing quality was checked by FastQC program (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Phred values were calculated. Only reads with Phred > 20 were used. Adaptor sequences were removed by the cutadapt program [47 ] and reads were shorter than 17 nt and longer than 36 nt were removed by BBDuk. Reads were mapped on satellite consensus dimer sequences using the bowtie2 program [48 (link)] and FPKM values were obtained.

Small RNAs extracted from RNA-IPs were migrated on a 1.5% agarose gel and recovered from the corresponding bands using the Monarch DNA Gel Extraction kit (NEB #T1020 New England Biolab). RNA samples were used to prepare libraries using the NEBNext Multiplex Small RNA Library Prep Set (NEB #E7300S New England Biolab). The final PCR enrichment was performed using 15 cycles. Samples were quantified with Qubit and Agilent Bioanalyzer using the DNA high-sensitivity assays and sequenced on a NextSeq550 machine at the CSHL Genome Center.

Total RNA (TNA) from symptomatic and asymptomatic leaves was extracted using a hexadecyltrimethylammonium bromide (CTAB)-based method. A TNA aliquot (3 μg) was separated by PAGE (Cao et al., 2014 (link)) and, using the 14–30 ssRNA Ladder Marker (TAKARA, Japan) as molecular marker, small RNAs with a size between 14 and 30 nt were recovered from the gel. The sRNA library was prepared with a NEBNext^®Multiplex Small RNA Library Prep Set (New England BioLabs^®Inc., United States) and sequenced using the BGISEQ-500RS (Huada Gene, Shenzhen, China). Raw reads data for the symptomatic and asymptomatic samples were processed to trim the adaptors and low-quality reads (quality score limit = 0.05; maximum number of ambiguities = 2; reads below 18 and above 30 nt discarded) were filtered out (Zhang et al., 2018 (link)). High quality reads were further filtered to remove host reads by mapping them to the Ziziphus jujuba (common jujube) genome² (Liu et al., 2014 (link)). Retained reads were assembled de novo into larger overlapping sequences (contigs) using Velvet software 0.7.3 (Zerbino, 2010 (link)). Output sRNA contigs were used to search for similar sequences available in NCBI databases using BLAST (x and n) analysis³.

A total of 48 RNA samples from head kidney were sequenced. There were 8 samples from each of the time points T1–T6. The small-RNA libraries for the 48 samples were constructed by use of NEBnext^® multiplex small RNA Library Prep Set (New England Biolabs, Inc., Ipswich, MA, USA) in accordance with manufacturers protocol. One µg of total RNA from each of the samples was used as input for preparation of the libraries that included 5′ and 3′ adapter ligation, reverse transcription, PCR amplification and size selection of 140–150 bp fragments using 6% polyacrylamide gel. The sequencing was performed on an Illumina NextSeq 500 providing 75 bp single end reads. Both library preparation and sequencing were performed at the Norwegian Sequencing Centre (NSC). All sequenced samples have been submitted to the NCBI Sequence Read Archieve (SRA) (https://www.ncbi.nlm.nih.gov/sra/) with accession bioproject number PRJNA556577.