After US + nano-se@PC treatments, S. cerevisiae were separated by centrifugation (5000 rpm, 4 min). The total RNA of S. cerevisiae was extracted using RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol, and treated with RNase-free DNase Ⅰ (Takara) to remove contaminating genomic DNA. Then, RNAs were quantified at 260 nm, and the integrity of RNAs was evaluated by formaldehyde agarose gel electrophoresis. RNA-Seq profiling were conducted by BGI (Beijing Genomics Institute, Shenzhen, China). Briefly, the mRNA was separated from the total RNA and short fragments were obtained by adding fragmentation buffer. The cDNA was obtained by reverse transcription using random hexamer-primers, followed by the synthesis of the second cDNA strand using DNA polymerase Ⅰ and RNase H. After purification and amplification, the cDNA library was created and sequenced, respectively, on the Illumina Hi-Seq 2000 platform. The raw data were filtered via deleting the adaptor and low-quality sequences. The differentially expressed genes (DEGs) were defined based on the false discovery rate (FDR) < 0.05 and log2 (fold change) ≥ 1. The enrichment analysis of DEGs were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and Bonferroni-corrected p < 0.05 was considered statistically significant.
Rnase free dnase 1
Manufactured by Takara Bio
Sourced in Japan, China, United States, Germany
RNase-free DNase I is an enzyme that selectively degrades DNA in the presence of RNA. It is commonly used in molecular biology applications to remove contaminating DNA from RNA samples, ensuring the purity of RNA for downstream analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (201 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (69 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (69 mentions)
Primescript rt reagent kit, by Takara Bio (36 mentions)
Trizol, by Thermo Fisher Scientific (33 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (28 mentions)
M mlv reverse transcriptase, by Promega (27 mentions)
2100 bioanalyzer, by Agilent Technologies (26 mentions)
Rneasy mini kit, by Qiagen (24 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (24 mentions)
Rnaiso plus, by Takara Bio (21 mentions)
Sybr premix ex taq, by Takara Bio (18 mentions)
Hiseq 2000 platform, by Illumina (17 mentions)
Sybr premix ex taq 2, by Takara Bio (15 mentions)
Trizol kit, by Promega (14 mentions)
M mlv reverse transcriptase, by Takara Bio (14 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (14 mentions)
Sybr premix ex taq kit, by Takara Bio (13 mentions)
Hiseq 2500 platform, by Illumina (13 mentions)
Hiseq 2500, by Illumina (13 mentions)
658 protocols using rnase free dnase 1
1
Transcriptional Profiling of S. cerevisiae
2
RNA Extraction and Sequencing
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Total RNA of WT and IRL were extracted using Trizol reagent, and the crude extracts were treated with RNase-free DNaseⅠ(TaKaRa, Dalian, China). In order to reduce the individual error, the RNA of 3 independent strains of RBSDV infected or wild type plants were mixed as one sample. The total RNA concentration was examined with a spectrophotometer (Nanodrop ND-2000, ThermoFisher Scientific, Wilmington, DE, USA), and the RNA sample integrity was verified by a Bio-Analyzer 2100 (Agilent Technologies, Santa.Clara, USA). The cDNA libraries construction and Sequencing at BGI Tech Solutions Co., Ltd. (BGI Tech, Shenzhen, China).
3
Extraction of Total RNA from Lymphocytes
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Total RNA was extracted from the lymphocytes of C. auratus using the Trizol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. DNA contamination was removed by RNase-free DNase 1 (Takara, Dalian, China) treatment. The quantity of total RNA was estimated by agarose gel electrophoresis and UV spectrophotometry (Implen GmbH, Munich, Germany).
4
Comprehensive RNA Extraction from I. sinensis
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Total RNA was extracted from leaf tissues of I. sinensis. Samples were collected from four accessions cultivated at Wuhan Botanical Garden, and were immediately frozen in liquid nitrogen. One milliliter of TRIzol reagent (Invitrogen, CA, USA) was added for every 100 mg leaf tissue and treated with RNase-free DNase 1 (TaKaRa Bio, Shandong, China) for 1 h at 37°C. RNA was then dissolved in RNase-free water (Ambion, USA). A 1-µL aliquot of each sample was used to check RNA quality and concentration with NanoDrop 2000 Spectrophotometer (Thermo Fischer Scientific Inc., DE, USA) and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). To maximize the quality of the transcriptional units and improve the analytical process, a pool was created by mixing equal volumes of total RNA from each sample.
5
Transcriptome Profiling of Leaf, Stem, and Root Tissues
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A mix of tissues from leaves, stems and roots were collected at 12 am and 12 pm. One individual for each species was sampled, as the intra-species variation is low compared with the inter-species variation50 and life-form of the three species is stable. Total RNA was isolated using RNAisoTM Plus (Takara, Qingdao, China) and then treated with RNase-free DNase I (Takara, Qingdao, China) for 45 min according to the manufacturer’s protocols. The quality of total RNA was checked using 2% agarose gel electrophoresis. The RNA samples were then delivered to Beijing Genomics Institute (BGI, Shenzhen, China), and concentrations were checked by Agilent Technologies 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara CA, USA). The cDNA preparation and Illumina sequencing were performed at BGI. The entire process followed a standardized procedure monitored by BGI’s Quality Control System. The mRNA was isolated from total RNA using oligo (dT) magnetic beads using the manufacturer’s instructions for cDNA library construction. Double stranded cDNA was sequenced using the Illumina HiSeq™ 2000 sequencer (90 bp paired-end). Image data from the sequencer was transformed by base calling into raw sequence data, which formed the raw reads.
6
Tiller Bud RNA Extraction and RACE
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Total RNA was extracted from the tiller buds of the Tin1/tin1 NIL plants at 25 DAP, treated with the RNase-free DNase I (Takara) and purified using the RNAclean kit (Tiangen). 5′ and 3′ RACE were carried out using the SMART RACE cDNA Amplification Kit (Clontech) following the manufacturer’s instructions.
7
Total RNA Extraction and Transcriptome Sequencing
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Total RNA was extracted using the total RNA kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase I (Takara, Dalian, China) to avoid DNA contamination. Agarose gel electrophoresis (1.5%), a Nanodrop 2000 (Thermo Scientific, Wilmington, NC, USA), and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) were used to check the integrity, concentration, and purity of RNA. Library preparation was performed using the NEBNext® Ultra™ RNALibrary Prep Kit for Illumina® (NEB, Ipswich, MA, USA) following the manufacturer’s instructions. The libraries were sequenced on an Illumina HiSeq 2000 platform at the Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). All raw data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (No. PRJNA596893 ).
8
Total RNA Extraction and Quality Assessment
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Total RNA was extracted using the TRIzol method (Invitrogen, CA, USA) and treated with RNase-free DNase I (Takara, Kusatsu, Japan). RNA degradation and contamination was monitored on 1% agarose gels. RNA concentration and purity was measured using NanoDrop spectrophotometer (Thermo Scientific, DE, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA).
9
Biotin-labeled RNA Pulldown for circPTK2
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Biotin-labeled RNA for liner sequence of circPTK2 was generated by an in vitro transcription reaction with the Biotin RNA Labeling Mix (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche, Mannheim, Germany), and then treated with RNase-free DNase I (Takara, Japan). After incubation with guide oligonucleotide targeting circular junction, the liner probe was then circularized using T4 RNA ligase I, treated with RNase R. After purified with RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), the biotin-labeled RNA probe (3 μg) was then incubated with cell extracts from CRC cells at room temperature (RT) for 2 h, and treated with 35 μl of Streptavidin C1 magnetic beads (Invitrogen) for 1 h. after washed, the retrieved protein was detected by western blot or mass spectrometry analysis (CapitalBio Technology, Beijing, China).
10
Isolation and Sequencing of miRNA
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Total RNA was isolated from each group from spleen samples at a 24 h time point using the Trizol kit (Invitrogen, Carlsbad, CA, USA), as directed by the manufacturer. Genomic DNA contamination was removed using RNase-free DNase I (TaKaRa, Osaka, Japan). RNA integrity was evaluated via agarose gel electrophoresis. The quality and concentration of the RNA were measured using a spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA), and the completeness of the RNA was assessed by employing a bioanalyzer (Agilent Bioanalyzer 2100, Pleasanton, CA, USA). Then, the TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA) were used to prepare the miRNA sequencing library. Finally, the constructed library was sequenced using Illumina Hiseq 2000/2500 (Illumina, San Diego, CA, USA), and a single-ended reading code of 50 bp was sequenced.
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