Hiseq x ten instrument

Metaphase II oocytes after NMN treatment were collected from control, heat-stressed and NMN-supplemented groups (100 oocytes per group), and total RNA was extracted using RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. Extracted RNA was quantified with the Qubit RNA Assay Kit (ThermoFisher Scientific). mRNA library construction was performed with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manuals. The protocol consisted of sequential RNA fragmentation, reverse transcription using random primers, second strand cDNA synthesis, end repair, dA-tailing, adapter ligation, and PCR enrichment. The concentration and quality of libraries were tested by a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific), qPCR, and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Then the libraries were sequenced on Illumina Hiseq X Ten instruments with 150 bp pair-end reads. High-quality sequences (clean reads) were obtained by trimming adaptor sequences and removing low-quality reads from raw RNA-seq reads using cutadapt (v1.10). Reads were then aligned to the susScr11 reference genome using tophat2 (v2.0.13).

DNA was extracted from 10 g of sediment sample for each experiment replicate, using the MoBio PowerSoil DNA Isolation kit (MO BIO Laboratories, USA) according to the manufacturer’s protocol. DNA concentrations of the extract were measured with a Qubit fluorometer. For each library construction, about 100 ng DNA was fragmented with Covaris S2 (Covaris, USA) and was used to construct metagenomic DNA library with NEXTflex™ DNA Sequencing Kit compatible with the Biomek® FXp (Bio Scientific, USA). Notably, PCR amplification was limited to 12 cycles for each Illumina library. The quality of DNA library was examined by Agilent Bioanalyzer 2100 (Agilent, USA) with DNA 12000 Kit. Paired-end Illumina sequencing (2 × 150 bp) was performed for each metagenomic library on Hiseq Xten instruments (Illumina).

Following resection, a representative tumor fragment was isolated and transferred rapidly to the laboratory for study as previously described [20 (link)]. Briefly, tissue was initially cut into segments and subjected to digestion by collagenase type I/II (Thermo Fisher Scientific, USA) and DNAse I (Sigma, USA). The digested pieces were triturated with a 1 ml syringe plunger and passed through a 70 μm cell strainer (Coring, USA). After resuspending in red blood cell lysis buffer (Solarbio, China), live cells were enriched using a Dead Cell Removal kit (Miltenyi Biotec, Germany) as per manufacturer’s instructions. Enriched live cells were washed and counted using a hemocytometer with trypan blue. Cells were then resuspended in PBS containing 0.04% BSA at a concentration of 1 × 10⁶ cells/ml with a viability of > 80% as determined with the Countess. Overall, the entire dissociation procedure took about 2 h from obtaining sample to generating single-cell suspension. The single-cell suspension was then run on the Chromium 10X device (10 × Genomics, USA).
Single-cell library preparation was carried out using Chromium Single cell 3’ Reagent v2 Kits (10 × Genomics, USA) according to the manufacturer’s protocol. Then the library was sequenced on the HiSeq X Ten instruments (Illumina, USA) and 150 bp paired-end reads were generated.

Trizol was used to extract total RNA based on provided directions, after which a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to measure RNA concentrations and purity, while an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) was utilized to measure the integrity of isolated RNA. A TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, CA, USA) was then used based on provided directions to prepare a sequencing library. All sequencing and analyses were performed by OE Bioinformatics Technology Co., Ltd. (Shanghai, China).
An Illumina HiSeq X Ten instrument was used to conduct 150 bp paired-end sequencing. Raw fastq data filed were initially processed with Trimmomatic, and clean reads were obtained via the removal of those reads considered of low quality. These clean reads were then aligned to the GRCh38 human reference genome with HISAT2, after which Cufflinks was used to calculate FPKM values for individual genes, while HTSeq-count was used to generate read counts. R was used to identify differentially expressed transcripts (P<0.05 and FC>2 or FC<0.5). Hierarchical clustering analyses of identified differentially expressed transcripts were then performed, after which GO and KEGG enrichment analyses of differentially expressed genes were conducted based upon hypergeometric distributions with R.

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.

Total RNA of each sample was extracted using TRIzol Reagent. RNA quality was quantified and qualified by Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA (1 μg) with RNA integrity number (RIN) value above or equal 8 was used for following library preparation. Library was made using Illumina NEBNext® Ultra™ Directional RNA Library Prep Kit (E7420L, NEB) according to the manufacturer’s protocol and the rRNA was depleted from total RNA using Ribo-Zero™ rRNA removal Kit. The libraries were loaded on an Illumina HiSeq X ten instrument accordingthe to manufacturer’s instructions (Illumina). Sequencing was carried out using a 2 × 150 paired-end configuration, image analysis and base calling were conducted by the HiSeq Control Software (HCS) (v3.3.76.1) + OLB (v1.9.3) + GAPipeline-1.6 (Illumina) on the HiSeq instrument. Computation analysis of paired-end reads was conducted using cutadapt (v1.15), Samtools (v0.1.19), Hisat2 (v2.1.0), and HT-seq (v0.11.2) software. Statistical normalization and differential analysis were performed in R using DESeq2 (v1.24.0) package. The threshold to define up or down regulation was fold-change > 1.5 and FDR < 0.05. Visualization were also conducted in R (v3.3.3).