Library preparation, target enrichment and sequencing of the cfDNA samples was performed according to the standard operating procedures at Personalis. Briefly, pre-enrichment libraries were prepared from 2 ng to 30 ng cfDNA input using a commercially available KAPA Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) and Personalis-optimized workflows. A Lunatic spectrophotometer (Unchained Labs, Pleasanton, CA, USA) was used to quantify the pre-enrichment libraries. Up to 1500 ng of library DNA was enriched with NeXT Personal custom probe panels using proprietary modifications to a commercially available Twist kit and hybridization-capture workflow (Twist Bioscience, South San Francisco, CA, USA). The post-enrichment libraries were significantly over-sequenced on NovaSeq instruments (Illumina, San Diego, CA, USA) in order to optimize the number of unique molecules observed.
Novaseq instrument
Manufactured by Illumina
Sourced in United States, China
The NovaSeq instrument is a high-throughput gene sequencing system developed by Illumina. It is designed for large-scale genomic analysis, enabling rapid and efficient DNA sequencing. The NovaSeq instrument can generate a large volume of sequencing data in a single run, making it suitable for various applications such as whole-genome sequencing, transcriptome analysis, and targeted sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq flow cell, by Illumina (5 mentions)
Novaseq s1 reagent kit, by Illumina (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Kapa library quant kit, by Roche (3 mentions)
Protein lobind tube, by Eppendorf (2 mentions)
Sodium butyrate, by Merck Group (2 mentions)
Ab4729, by Abcam (2 mentions)
Ab8580, by Cell Signaling Technology (2 mentions)
Ptm 1406, by Abcam (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Truseq mrna library preparation kit, by Illumina (2 mentions)
Library quant kit, by Roche (2 mentions)
Nextera xt kit, by Illumina (2 mentions)
Nebnext rrna depletion kit, by New England Biolabs (2 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Chromium controller, by 10x Genomics (2 mentions)
88 protocols using novaseq instrument
1
Personalis cfDNA Sequencing Protocol
2
CUT&Tag Profiling of Histone Modifications
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CUT&Tag was performed as described earlier starting from nuclei [28 (link),29 (link)]. First, nuclei were isolated from the sorted macrophages. In short, the macrophages were centrifuged for 5 min at 4 °C, 500 rpm, supernatant was removed, and the cells were lysed on ice in 1 mL of nucleus extraction buffer (1 × prelysis buffer from the EpiGentek EpiQuick Total Histone Extraction Kit, OP-0006-100). To stop the lysis reaction, 1 mL of PBS+1%BSA was added, and nuclei were collected through centrifugation for 5 min at 4 °C, 500 rpm. The supernatant was removed, the nuclei were resuspended in PBS+1% BSA, and a sample was visually inspected for viability, purity, and abundance of nuclei under the microscope. Next, CUT&Tag libraries were created according to the published CUT&Tag protocol for nuclei [29 (link)]. All buffers were supplemented with 5 mM sodium-butyrate (Sigma, 303410) and 1X complete protease inhibitor (Merck, 1187358000). Protein lo-bind tubes (Eppendorf, EP0030108116) were used to reduce sample loss. Antibodies against H3K18la (PTM-Bio, PTM-1406), H3K4me3 (Abcam, ab8580), H3K27me3 (Cell Signaling Technology, C36B11) and H3K27ac (Abcam, ab4729) were used in this study. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.
3
CUT&Tag for Histone Modifications
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CUT&Tag was performed according to the published CUT&Tag protocol [29 (link)] for nuclei (BMDM, PIM, MB, MT, GAS, ADIPO) or cells (ESC). All buffers were supplemented with 5 mM sodium-butyrate (Sigma, 303410) and 1X complete protease inhibitor (Merck, 1187358000). Protein lo-bind tubes (Eppendorf, EP0030108116) were used to reduce sample loss. For GAS samples, incubation volumes were doubled to account for the tissue debris that remained in the nuclear suspensions since this gave better Tapestation QC results. Antibodies against H3K18la (PTM-Bio, PTM-1406), H3K4me3 (Abcam, ab8580), H3K27me3 (Cell Signaling Technology, C36B11), H3K27ac (Abcam, ab4729), and H3K9me3 (Abcam, ab8898) were used in this study. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.
4
Total RNA Extraction and RNA-Seq Library Preparation
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According to the manufacturer’s protocol, total RNA was extracted using the RNeasy kit with on-column DNase treatment (74004; Qiagen). RNA quality was assessed using an Agilent TapeStation (Agilent, Palo Alto, CA, USA), and RNA concentration was quantified by Qubit 4.0 spectrophotometer. Total RNA was used as the input material for rRNA depletion and RNA sequencing library preparation. The library was prepared for sequencing using the TruSeq standard total RNA library preparation kit (Illumina, San Diego, CA, USA). Each sample was prepared using 300 ng of total RNA. The resulting cDNA libraries were quantified using the KAPA library quantification kit (KAPA Biosystems, Wilmington, MA, USA), and quality and size were checked using the Agilent high-sensitivity DNA kit. The average fragment length of the cDNA libraries was 330 bp. RNA-Seq libraries were sequenced using paired-end 100-base (PE100) sequencing chemistry on NovaSeq instruments following the manufacturer’s protocols (Illumina, San Diego, CA, USA). RNA sequencing was performed at the sequencing core facility (Core Technologies Research Initiative [CoTeRI]) of the National Institute of Biomedical Genomics (NIBMG) in Kalyani, India.
5
Transcriptional Profiling of PPARγ Knockdown
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PDLF-1 were transfected with si-PPARγ or si-control for 24 h. These cells were then cultured in induction medium for 6 days and total RNA was purified before and after the culture. Purified RNA was DNase-treated and used in RT-qPCR and RNA-seq analyses. Input RNA was converted to cDNA libraries using the NEBNExt poly (A) mRNA magnetic isolation module. Libraries were sequenced using 150-bp paired-end reads on Illumina Novaseq instruments. Library preparation and RNA-seq were performed by Novogene (Beijing, China).
Adapter trimming was conducted with Trim galore version 0.6.6 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ (accessed on 3 May 2021)) with default settings and then aligned to a reference genome (hg38) using HISAT2 version 2.2.1 [63 (link)]. Tag directories were generated using ‘makeTagDirectory’ in HOMER [64 (link)] and gene expression at exons was quantified with the ‘analyzeRepeast.pl’ command in HOMER using ‘-strand both’ and ‘-count exons’ to identify expressed genes.
Adapter trimming was conducted with Trim galore version 0.6.6 (
6
RNA-seq analysis of mouse transcriptome
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RNA-seq was performed according to our previous study [18 (link)]. For the purpose of constructing the sequencing libraries, a total of 1 μg RNA was obtained from each sample. We converted the RNA into double-stranded cDNA in accordance with the reverse transcription kit instructions, and then we digested and labeled the double-stranded cDNA using Tn5 transposase. The final step was enrichment PCR using the HiFi PCR Mix for NGS. An Agilent 2100 Bioanalyzer was used to quantify the libraries. Using an Illumina NovaSeq instrument, paired-end sequencing of the library was conducted (sequencing was performed by GENEWIZ Biotech). A STAR program was used to map the reads to the mouse genome (http://code.google.com/p/rna-star/ ). Genes that showed a 1.5-fold change in expression (P < 0.05) were considered significantly differentially expressed. Differentially expressed genes (DEGs) were enriched by Gene Ontology (GO) using Metascape (http://metascape.org ) [19 (link)].
7
Osteoblast Spheroid RNA-seq Protocol
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Osteoblast spheroids from the ground experiments were collected in RNAlater and frozen at −80°C for RNA collection. Total RNA was isolated with the miRNeasy Micro kit (Qiagen) following manufacturer instructions. Libraries were prepared using the SMART-seq mRNA kit28 (link),38 (link) (Takara Bio) and sequenced on NovaSeq instrument (Illumina) using 151bp paired-end reads. The reads were demultiplexed into FASTQ files using the BCL Convert Conversion Software (Illumina, v4) and the adapters were trimmed using Cutadapt v2.3 39 . The reads were then mapped to the human GRCh38 reference genome (ENSEMBL 109) following ENCODE standards using STAR v2.7.8a 40 (link) and counts were assigned to genes using RSEM v1.3.3. The final libraries contained an average of 18,095,188 uniquely mapped reads.
8
SARS-CoV-2 Variant Identification Protocol
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Participants were tested for SARS‐CoV‐2 by reverse‐transcription polymerase chain reaction tests using respiratory specimens collected for clinical or research purposes. We classified participants with a positive SARS‐CoV‐2 result as cases. Participants who had discordant clinical and research results were categorized as a case if at least one of the results were positive. We classified participants with only negative SARS‐CoV‐2 results as controls.
In addition, SARS‐CoV‐2 virus variants from a subset of SARS‐CoV‐2 positive participants with onset dates between November 9, 2021, and January 9, 2022, were identified by whole genome sequencing. Research‐collected SARS‐CoV‐2 positive respiratory specimens with cycle threshold values <30 and stored in appropriate transport medium were prepared for sequencing using the xGen SARS‐CoV‐2 library preparation kit (Integrated DNA Technologies, Inc., Coralville, IA). Libraries were sequenced on a NovaSeq instrument (Illumina Inc., San Diego, CA). A single consensus genome for each sample was generated. SARS‐CoV‐2 variants were determined using Pangolin version 3.1.20 (pangoLEARN 1.2.123, Scorpio 0.3.16).9
In addition, SARS‐CoV‐2 virus variants from a subset of SARS‐CoV‐2 positive participants with onset dates between November 9, 2021, and January 9, 2022, were identified by whole genome sequencing. Research‐collected SARS‐CoV‐2 positive respiratory specimens with cycle threshold values <30 and stored in appropriate transport medium were prepared for sequencing using the xGen SARS‐CoV‐2 library preparation kit (Integrated DNA Technologies, Inc., Coralville, IA). Libraries were sequenced on a NovaSeq instrument (Illumina Inc., San Diego, CA). A single consensus genome for each sample was generated. SARS‐CoV‐2 variants were determined using Pangolin version 3.1.20 (pangoLEARN 1.2.123, Scorpio 0.3.16).
9
RNA-Seq Library Preparation and Sequencing
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RNA samples were run on a Bioanalyzer (Agilent) at a concentration of 1.5 ng/μl using the eukaryote total RNA pico series II assay (Agilent) to assess RNA integrity. Libraries for RNA sequencing were prepared from 5 ng RNA/sample using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) according to the manufacturer’s instructions using 12 PCR cycles for amplification. DNA concentration of libraries was determined using Qubit dsDNA HS Assay Kit, and libraries were diluted to 1.5 ng/μl, then run on a Bioanalyzer (Agilent) using the High Sensitivity DNA Assay Protocol (Agilent) for quality control. Libraries were sequenced on an Illumina NovaSeq instrument, single-end at 100 bp.
10
Single-Cell Library Construction Using BD Rhapsody
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Microbead-captured single-cell library construction was performed using a BD Rhapsody™ Single-Cell Analysis System (#633701, BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions as described previously.[8 (link)] The constructed libraries were sequenced on a NovaSeq instrument (Illumina, San Diego, CA, USA).
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