Paired end sample preparation kit

Targeted Sequencing was performed with the illumina Hiseq Xten platform at the sequencing laboratory of Tissuebank Precision Medical Co, Ltd. (Shanghai, China). A total of 10 ng DNA per sample was amplified by PCR, and then the library was captured by using xGen^® Lockdown^® probes and xGen Hybridization and Wash Kit; Illumina Hiseq sequencer carried out pair end sequencing with a depth of 200X. 43 pathogenic genes (ASXL1, BCOR, BCORL1, BRAF, CALR, CBL, CDKN2A, CEBPA, CREBBP, CSF3R, CUX1, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, GNAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KIT, KRAS, MPL, NF1, NPM1, NRAS, PHF6, PIGA, PTEN, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2) were screened in all patients, including the entire coding regions and exon-intron boundaries. This multi-gene panel was expected to cover 100% of the targeted area. DNA was sheared into short genetic fragments (150∼200 bp) using the Covaris LE220, which included purified and captured gene fragments. Adaptor-ligated amplicons were prepared using the Illumina Paired-End Sample Preparation kit. Illumina multi-PE-adaptors were bound to terminal genes and target enrichment was performed by probe capture, amplicons were purified using VAHTS DNA Clean Beads and captured on the Illumina Hiseq Xten instrument.

Genomic DNA was isolated from overnight cultures using a Wizard Genomic DNA purification kit (Promega, Madison, WI) and quantified with PicoGreen (Molecular Probes, Eugene, OR). Sequencing libraries were prepared using the Illumina Paired-End Sample Preparation Kit according to the manufacturer's protocol but with the recommended modification of performing gel extraction incubations at room temperature [35] (link). Prior to sequencing, library quality and insert size were evaluated on an Agilent Bioanalyzer 2100 DNA 7500 chip. Libraries were sequenced at the Iowa State University DNA Facility on an Illumina GAIIx for either 75 cycles (strains SW114, 12939, 84-15995, H465, and D74) or 100 cycles (strains Nagasaki, MN-H, 29755, 174 and SW140). GenBank accession numbers for the genomes are given in Table 2 and Kuehn et al. [36] (link).

The samples of “long-skinny” morphotype of R. piscesae were obtained during Alvin dive 4243 from the deep-sea hydrothermal vent at Cathedral vent, Main Endeavor Field of the Juan de Fuca Ridge (47° 56’ N, 129° 05’ W, 2,181 m depth) on August 9, 2006. Genomic DNA (gDNA) was extracted from vestimentum muscle of the specimen using a standard phenol/chloroform extraction protocol and broken into random fragments for whole-genome shotgun (WGS) sequencing. Agarose gel electrophoresis was used to check the quality of the gDNA, and Qubit system was used to quantify the gDNA. Short-insert paired-end libraries (180 bp, 300 bp and 500 bp) were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, USA) according to the standard protocol, respectively. Large-insert mate-pair libraries (2 kb, 5 kb, 10 kb and 15 kb) were prepared following the Cre-lox recombination-based protocol [64 (link)]. All DNA libraries were subjected to paired-end sequencing on the Illumina Hiseq 2000 platform (Illumina). Muscle samples from vestimentum were also collected for constructing RNA sequencing (RNA-seq) library. Total RNA was extracted with TRIzol reagent (Molecular Research Center, USA). Paired-end library for RNA-seq was constructed using the Paired-End Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on the Illumina Hiseq 2000 platform (Illumina).

Samples of root, stem, leave, and fruit tissues from three individuals were collected for total RNA extraction. We extracted total RNA from each sample using the MiniBEST Plant RNA Extraction Kit (TaKaRa, China) following the manufacturer’s protocol. The cDNA was synthesized using RNA to cDNA EcoDry Premix (Clontech) with oligo (dT) primer. The paired-end library was constructed based on the Paired-End sample Preparation kit protocol (Illumina, USA). RNA sequencing was performed on the NextSeq 500 platform (Illumina, USA). For RNA-seq analysis, we first used the HISAT2 software package to map RNA-seq data of roots, stems, leaves, and fruits to the H. tibetana genome [61 (link)]. The RSEM software was used to calculate the gene expression level as fragments per kilobase per million (FPKM) [62 (link)]. We used DESeq2 to identify differential expression genes with an adjusted p-value ≤ 0.05.

The Illumina RNA-Seq library preparation protocol includes poly-A RNA isolation, RNA fragmentation, reverse transcription to cDNA using random primers, adapter ligation, size-selection from a gel and PCR enrichment [65 ]. The batch of libraries was sequenced at the BYU sequencing center and by Otogenetics (Norcross, GA) using Illumina Hi-seq 2000 sequencer. This includes cDNA libraries of Suaeda 0 mM NaCl-treated shoot and roots in triplicates and cDNA libraries of Suaeda 300 mM NaCl-treated shoot and roots in triplicates. The paired-end library was developed according to the protocol of the Paired-End sample Preparation kit (Illumina, USA).

Total RNAs were extracted from three replicates group of 3 days old virgin females of w¹¹¹⁸ and dIlp8^−/− stocks. The quantity and quality of RNA have been assessed by Nanodrop, Qubit 2.0 and Agilent 2100. Magnetic beads with oligo (dT) were performed to enrich Poly (A) mRNA from total RNA. The cDNA library was constructed using the Paired-End Sample Preparation Kit (Illumina Inc., San Diego, CA, United States) followed the manufacturer’s instructions. To ensure quality control, Qubit 2.0, Agilent 2100 and quantitative Real-Time PCR were performed, and then the transcriptome sequencing was carried out using IlluminaHiSeq 6000 with PE100 approach (Biomarker Technology Corporation, Beijing, China). The clean reads were filtered from raw data by removing adaptor sequence, primer reads and low-quality bases. We use the longest isoform to represent a gene for downstream analysis. The reads of each sample were mapped to the reference genome of D. melanogaster. The gene abundance was represented by PKM value. DEG screening in sample group was conducted with the DEGseq package. False discovery rate (FDR) value ≤ .01 and fold change (FC) ≥ 2 in the Benjamini and Hochberg method were chosen as DEGs.