Approximately 200 ng of genomic DNA from tumor tissues and matched germline controls (PBMCs) were subjected to ultrasonic fragmentation by Covaris S220 sonicator (Covaris, Woburn, MA, USA) to obtain a DNA fragment size ranging from 200 to 500 base pairs (bps). Fragmented genomic DNA was ligated to adapters containing sample specific indexing barcode (Illumina, San Diego, CA, USA) to construct a DNA library. Library construction was also performed for CTCs but the fragmentation was skipped because the amplicon size of WGA from CTCs was already in the desired range. After 8–12 cycles of polymerase chain reaction amplification, exonic region of input DNA samples were enriched by hybridization with Agilent SureSelect Human v6 probes (Agilent, Santa Clara, CA, USA). The enriched DNA were subjected to Illumina NovaSeq 4000 (Illumina, San Diego, CA, USA) for NGS with a 2 × 150 bp format. The average coverage depths of captured region were estimated 300× on tumor tissues, and 100× on CTCs and germline controls.
Novaseq 4000
Manufactured by Illumina
Sourced in United States
The NovaSeq 4000 is a high-throughput DNA sequencing system designed for large-scale genomic research. It utilizes advanced sequencing-by-synthesis technology to generate high-quality DNA sequence data. The NovaSeq 4000 is capable of processing multiple samples simultaneously, delivering increased throughput and efficiency for genomic applications.
Automatically generated - may contain errors
Lab products found in correlation
S220 sonicator, by Covaris (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Axyprep multisource rna miniprep kit, by Corning (1 mentions)
Paxgene blood rna kit, by Qiagen (1 mentions)
Fastqc, by Babraham Bioinformatics (1 mentions)
Fragmentation buffer, by Illumina (1 mentions)
Sequencing adaptor, by Illumina (1 mentions)
Oligo dt beads, by Illumina (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Dna polymerase 1, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
7 protocols using novaseq 4000
1
Targeted Exome Sequencing of Tumor and CTC
2
Comprehensive RNA-seq Workflow for Transcriptome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction and quality assessment, cDNA libraries preparation, data assembly, sequence alignment of reference genomes, and unigene annotation were performed by Gene Denovo Biotechnology Co. (Guangzhou, China). Following the manufacturer’s instructions, total RNA was extracted from each sample using Trizol reagent (Invitrogen, MA, USA). The cDNA libraries were sequenced using an Illumina NovaSeq™ 4000 instrument that generated paired-end reads lengths of 200 bp, and the clean reads were aligned to the C. higginsianum or A. thaliana genome assembly with HISAT2 [43 (link)]. Stringtie was used to reconstruct the transcripts and calculate the expression of all genes in each sample, presented as FPKM value (Supplementary Datas S1 and S2 ) [44 (link)].
3
Transcriptome analysis of A. oligospora response to nematodes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WT and Δfus3 strains were grown on cellophane-overlaid PDA plates at 28°C for 5 days. Approximately 600 nematodes were then added to each plate for 0, 12, 24, and 36 h of induction, and three replicates were used for each sample. After collecting the samples, RNA was extracted with the AxyPrep multisource RNA miniprep Kit (Axygen, Jiangsu, China), and then sent to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for transcriptome sequencing. The final cDNA library was sequenced on an Illumina Novaseq 4000 platform. The clean reads were mapped to the A. oligospora (ATCC 24927) genome sequence. The gene expression changes were evaluated and the DEGs were identified with an FDR value of ≤0.05. High-throughput sequencing data were analyzed using the OmicShare online platform (www.majorbio.com ).
4
Transcriptomic Analysis of YTHDC2 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA sequencing was performed on leukocytes obtained from the 3 patients with YTHDC2 variants and from 7 female “controls” with POI of no known cause. RNA was extracted using the PAXgene Blood RNA kit (Qiagen). Libraries were sequenced with the NovaSeq 4000 (SP, 2 × 151 bp, Illumina). Reads underwent quality control (FastQC, Babraham Bioinformatics) and were aligned against the human reference genome sequence (NCBI, GRCh38) (STAR 2.5.2a) (72 (link)). Fastp, DESeq2, DEXSeq, and TEtranscripts were used for preprocessing, differential gene expression analysis, differential exon usage/splicing, and differential transposable element expression, respectively (74 (link), 78 (link)–80 (link)). Downstream bioinformatic analysis was undertaken as described for fetal samples. Differentially expressed genes of interest were verified using qRT-PCR (TRIM9: Hs00364838_m1; SYCP2L: Hs00293769_m1; Applied Biosystems).
5
Single-Cell TCR and Transcriptome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T-cell receptor (TCR) sequencing and 5’ gene expression sequencing was performed using the Chromium Controller and Single Cell 5’ Library & Gel Bead Kit (10x Genomics) according to the manufacturer’s protocol. Sequencing was performed using the Illumina NovaSeq 4000 platform and the 150bp paired-end configuration.
6
Illumina Sequencing Library Construction and Quality Control
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Library construction and sequencing services (paired-end 150 bp using an Illumina NovaSeq 4000) were carried out by Novogene Corporation, Inc. (Sacramento, CA, USA). Briefly, for library construction, RNA samples were enriched using oligo(dT) beads (Illumina, San Diego, CA, USA). Then, the mRNA was fragmented randomly using a fragmentation buffer (Illumina), followed by the first strand cDNA synthesis using mRNA as a template, random hexamer primers, a custom buffer (Illumina), dNTPs (Invitrogen) and DNA polymerase I (Invitrogen). The second strand was synthesized after a RNAse H treatment (Invitrogen). Finally, after the sequence terminal repair, sequencing adaptors were ligated (Illumina). The double-stranded cDNA library was completed through size selection and PCR enrichment.
FastQC v0.11.5 software tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) was used to assess read quality and detect Illumina adaptors. Subsequently, Trimmomatic v0.36 [89 (link)] was used to trim off low-quality bases at the 5′ and 3´ends using the parameters as follows: trailing: 5; leading: 5; and sliding-window 4:15. Reads shorter than 50 bp and adaptors were removed using the parameters ILLUMINACLIP: TruSeq3-PE-2.fa:2:30:10.
FastQC v0.11.5 software tool (
7
Multigenerational Fish Family Sampling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We selected three families from a previous laboratory experiment (Morris et al. 2020 (link)), each of which contains one father, one mother, five sons, and five daughters. During the experiment setup, all the fish were kept in the same flow-through aquarium rack, and experienced the same feeding and temperature regimes in the laboratory, as well as the same water within the cell flow system. All the fathers and mothers were paired when they were 4 mo old. We sampled offspring when they were sexually mature, which is roughly 4 mo of age, from two consecutive clutches. In total, 36 samples from three pedigrees were collected. DNA was extracted from whole head tissues with Qiagen DNeasy Blood & Tissue Kit, following the manufacturer protocol. We prepared the shotgun library with IDT dual-index adaptor. After DNA quality control, all the samples were sequenced individually on the Illumina NovaSeq 4000 platform.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!