Total RNA was extracted from the 7-week-old plants (remove root), RNA concentration was quantified using Qubit® 3.0 Flurometer (Life Technologies, CA, USA) and RNA integrity was assessed using RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The cDNA library construction and sequencing were performed by Annoroad Genomics (Beijing, China) on the Illumina Hiseq Xten platform (Illumina, San Diego, CA, USA). The mRNA of two 7-week-old plants (remove root) from WT or eno2− were sequenced as a biological relicate and three independent biological replicates were employed. The RNA-seq reads were aligned to Arabidopsis thaliana reference genome (TAIR 10.37) using HISAT2 (Sirén et al., 2014 (link)) and mapping rate were over 95%. Gene expression levels were assessed using Fragments per Kilobase per Million Mapped Fragments (FPKM).
Qubit 3.0 flurometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit® 3.0 Fluorometer is a compact and accurate instrument designed for the quantification of DNA, RNA, and protein samples. It utilizes fluorescence-based detection methods to provide precise and sensitive measurements of sample concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp system, by Beckman Coulter (8 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (6 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (5 mentions)
Qubit dna assay kit, by Thermo Fisher Scientific (4 mentions)
Nanophotometer, by Implen (4 mentions)
Novaseq 6000 platform, by Illumina (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Nanophotometer spectrophotometer, by Implen (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (3 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
2100 rna nano 6000 assay kit, by Agilent Technologies (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Idt xgen exome research panel v1, by Integrated DNA Technologies (2 mentions)
Magnetic universal genomic dna kit, by Tiangen Biotech (2 mentions)
Dnasecure plant kit, by Tiangen Biotech (2 mentions)
Pe150, by Illumina (2 mentions)
Hiseq x ten platform, by Illumina (1 mentions)
Qubit rna assay kit in, by Thermo Fisher Scientific (1 mentions)
34 protocols using qubit 3.0 flurometer
1
Transcriptome Analysis of Arabidopsis eno2 Mutant
2
Homo RNA-seq Transcriptome Analysis
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We collected roots, stems, leaves, and flowers of Homo to perform RNA-seq, and total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, California, USA). RNA concentration was measured using Qubit RNA Assay Kit in Qubit 3.0 Flurometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bio-analyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations. Total RNA (4 μg) with RNA integrity number (RIN) >7.5 was used as input material for library construction. The RNA-seq library was commercially performed using the DNBSEQ-T7 (DNBSEQ-T7, RRID:SCR_017981) with a PE read length of 150 bp at Annoroad Gene Technology Co. Ltd.
3
ChIP-seq Analysis of Neurospora crassa
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ChIP-seq samples were prepared as above described. DNA purity and concentration were measured using the NanoPhotometer® spectrophotometer (Implen) and Qubit® DNA Assay Kit in Qubit® 3.0 Flurometer (Life Technologies), respectively. Library construction was performed by Novogene Corporation (Beijing, China), and pair-end sequencing was performed on Illumina NovaSeq6000 platform. Raw reads that pass quality control were processed with Trim Galore (version 3.2) and mapped to N. crassa OR74A (NC10) genome using Bowtie2 (version 2.3.5.1) (43 (link)). Only unique mappers were retained for downstream analyses. Peak calling was performed using MACS2 (version 2.2.7.1) (44 (link)). Mapped read density was calculated over 10 bp bins and normalized by RPGC using bamCoverage from deepTools (version3.5.1) (45 (link)), denoised with sliding window (window size is 100 nt and step is 10 nt) and visualized with the Integrative Genomics Viewer (IGV) (46 (link)). The pipeline with detailed settings is available on GitHub (https://github.com/asuang/research-for-heterochromatin-in-NC ).
4
Viral DNA Extraction and TTV Detection
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Voided infant faeces were suspended in 1:20 (w/v) SM buffer, centrifuged twice at 4, 075 × g for 10 min at 4 °C, before filtering twice through 0.45 µm pore diameter filters. A 200 µL aliquot of the faecal viral-enriched suspension was lysed using a QIAGEN Blood and Tissue Purification Kit following the manufacturer’s recommendations with elution in 50 µL of TE Buffer. The concentration of double stranded viral DNA was calculated using a Qubit 3.0 Flurometer (Life Technologies, Carlsbad, CA, USA) using a Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher, Waltham, MA, USA). Subsequently, all dsDNA concentrations were normalised to 0.05 ng/µl. The choice of primers and conditions for detecting pan-human associated TTV by qPCR were as described by Ssemadaali et al. (2016) (link), using SensiFAST SYBR No-ROX mastermix and a LightCycler 480 thermocycler. A two-fold serial dilution of the purified TTV PCR product was also included in the qPCR for standard curve analysis.
5
Identify Off-Target CRISPR Editing Sites
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Genomic sequence containing the potential off-target sites identified by GUIDE-seq were PCR-amplified by 20 cycles with 2 × KAPA HiFi HotStart ReadyMix (Kapa Biosystems). Adapters containing the P5 and P7 sequences were incorporated into the PCR products by another 15 PCR cycles. The final Illumina libraries were purified with Ampure XP beads (Beckman) and quantified by Qubit 3.0 Flurometer (Life Technologies). Agilent 2100 Bioanalyzer (Agilent Technologies) was used to characterize the size and quality of the libraries. After Illumina sequencing, clean reads were extracted and aligned to the human genome hg19 using BWA. Samtools was used to count the variant reads number at each targeted sites.
6
RNA-seq analysis of adipocyte differentiation
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RNAs were collected from preadipocyte cells on Day(−2) and matured adipocytes Day(+4) and the amount and quality of RNAs were determined by NanoPhotometer®(IMPLEN, CA, USA), Qubit®3.0 Flurometer (Life Technologies, CA, USA) and 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA)10 (link). RNA-seq assays were performed on an Illumina Hiseq 2500 platform and clean reads were mapped to mouse genome (GRCm38/mm10) using HISAT2 v2.1.032 (link). Value of FPKM was calculated to represent the expression level of genes by HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html )33 (link).
7
Comprehensive RNA Extraction and Quality Assessment
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RNA extraction was applied with Trizol (Sigma-Aldrich, USA) traditional procedure. RNA degradation and contamination was detected by 1% agarose gels. RNA purity was checked using the kaiaoK5500® Spectrophotometer (Kaiao, Beijing, China). RNA concentration was tested by Qubit®3.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit on Bioanalyzer 2100 system (Agilent Technologies, USA).
8
Illumina MiSeq Sequencing of PCR Products
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Briefly, PCR products were purified and normalized using the SequalPrep Kit (Cat # A1051001; Thermo Fisher Scientific, Waltham, USA). PCR products were pooled in 10.0 µl volumes for each sample. Library prep was performed using the Illumina Nextera XT kit (Cat # FC-131-1096 and FC‐131‐1002; Illumina, San Diego, USA). Pooled fragments were assessed for correct size using the Agilent D5000 ScreenTape Station (Cat # G2940C, # 5067-4626, and # G2940CA; Agilent Technologies, Santa Clara, USA) and DNA concentration checked using the Qubit 3.0 Flurometer (Cat #Q33216 and #Q32853; Life Technologies Corporation, Carlsbad, USA). Sequencing was performed using the MiSeq v2 reagents using the 500 cycle kit (Cat # MS-102-2003; Illumina MiSeq reagents v2, San Diego, USA).
9
RNA-seq library preparation and sequencing
10
RNA Extraction and Sequencing Protocol
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Total RNA was extracted from pools of approximately 1000 guts using the Absolutely RNA Nanoprep kit (Agilent, USA) according to the manufacturer’s manual with slight modifications [63 (link)]. RNA quantity and quality were assessed using 1% agarose gels, Qubit® 3.0 Flurometer (Life Technologies, CA, USA) and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA (700 ng per sample) was used to generate adaptor-ligated double- stranded cDNA libraries for RNA-Seq using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (New England Biolabs, USA) following the manufacturer’s protocol. The fragment size and concentration of resultant libraries were carried out using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and StepOnePlus™ Real-Time PCR System (Library valid concentration > 10 nM).
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