Total RNA from muscle, liver, ileum and colon was extracted from 6 SBM and 6 RSM pigs using TRIzol protocol (Invitrogen) followed by RNeasy Plus Mini protocol (Qiagen). After the first washing step, on-column DNAse treatment was performed using PureLink DNase kit (Invitrogen). RNA purity and quality was measured using NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Only high quality (RIN ≥ 7) samples were sent for sequencing at the Norwegian Sequencing Centre (http://www.sequencing.uio.no ) and were used for validation of sequencing data by qRT-PCR. Six pigs from each dietary treatment were selected for RNA sequencing based on their FCR (three pigs with FCR higher than average and three pigs with FCR lower than average), while aiming at equal gender distribution. Pigs that were selected for sequencing originated from different litters in order to minimize the effect of single genotypes on the outcome of this study.
Purelink dnase kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PureLink DNase kit is a laboratory product designed for the removal of DNA from RNA samples. It provides a simple and efficient method to eliminate DNA contamination from RNA preparations, which is essential for accurate and sensitive downstream RNA analysis applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (2 mentions)
Purelink rna purification kit, by Thermo Fisher Scientific (2 mentions)
Precellys 24 homogenizer, by Bertin Technologies (2 mentions)
Reverseaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Purelink mini kit, by Thermo Fisher Scientific (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Trizol protocol, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Purelink micro to midi total rna purification system, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Mycycler thermal cycler, by Bio-Rad (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Power sybr green, by Thermo Fisher Scientific (1 mentions)
7500 fast qrt pcr system, by Thermo Fisher Scientific (1 mentions)
10 protocols using purelink dnase kit
1
Differential Gene Expression Analysis in Pigs
2
Transcriptomic Analysis of Unfertilized Eggs
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At all HPOs and HPSs, 1 g of the unfertilized eggs was placed in cryotubes, frozen in liquid nitrogen and then stored at -80°C freezer until RNA isolation. Samples were collected in three replicates for each fertilization time.
To prepare total RNA, an equal weight of 20 eggs was taken from each tube and treated with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) in Precellys24 homogenizer (Bertin Instruments), 5500 rpm for 2×20 sec with a 5 sec interval. RNA was isolated using the PureLink RNA Purification Kit (Invitrogen) according to the manufacturer’s instructions. During RNA isolation, samples were depleted of genomic DNA using a PureLink DNase Kit (Invitrogen) according to the manufacturer’s protocol. The RNA concentration and RNA purity were assessed using a NanoDrop Spectrophotometer (ND-1000, Thermo Scientific).
A concentration of 1000 ng RNA was used to generate cDNA using TaqMan reverse transcription Reagents (Life Technologies). Random hexamers were used to prime the reaction. Reverse transcription was performed at 25°C for 10 min, at 48°C for 1 hour and finally 95°C for 5 min. Control reactions were run without TaqMan reverse transcriptase and used as negative controls in the real-time PCR study.
To prepare total RNA, an equal weight of 20 eggs was taken from each tube and treated with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) in Precellys24 homogenizer (Bertin Instruments), 5500 rpm for 2×20 sec with a 5 sec interval. RNA was isolated using the PureLink RNA Purification Kit (Invitrogen) according to the manufacturer’s instructions. During RNA isolation, samples were depleted of genomic DNA using a PureLink DNase Kit (Invitrogen) according to the manufacturer’s protocol. The RNA concentration and RNA purity were assessed using a NanoDrop Spectrophotometer (ND-1000, Thermo Scientific).
A concentration of 1000 ng RNA was used to generate cDNA using TaqMan reverse transcription Reagents (Life Technologies). Random hexamers were used to prime the reaction. Reverse transcription was performed at 25°C for 10 min, at 48°C for 1 hour and finally 95°C for 5 min. Control reactions were run without TaqMan reverse transcriptase and used as negative controls in the real-time PCR study.
3
RNA Isolation from Unfertilized Eggs
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At all HPSs the unfertilized eggs (1 g, three replicates for each fertilization time point) were snap-frozen in liquid nitrogen prior storage at −80 °C until RNA isolation.
Total RNA was isolated from 20 eggs using TRIzol reagent (Invitrogen) in combination with PureLink RNA Purification Kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions. The eggs were homogenized in a Precellys24 homogenizer (Bertin Instruments), 5500 rpm for 2 × 20 sec with a 5 sec interval. The RNA was treated with DNaseI to avoid contatmination of genomic DNA using a PureLink DNase Kit (Invitrogen) according to the protocol. The concentration and purtity of the RNA were assessed using a NanoDrop Spectrophotometer (ND-1000, Thermo scientific). cDNA was synthezied from 1000 ng RNA using TaqMan reverse transcription Reagents (Life Technologies) and random hexamers to prime the reaction. Reverse transcription was performed at 25 °C for 10 min, 48 °C for 1 hour and 95 °C for 5 min. Reactions without TaqMan reverse transcriptase were used as negative controls in the real-time PCR study.
Total RNA was isolated from 20 eggs using TRIzol reagent (Invitrogen) in combination with PureLink RNA Purification Kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions. The eggs were homogenized in a Precellys24 homogenizer (Bertin Instruments), 5500 rpm for 2 × 20 sec with a 5 sec interval. The RNA was treated with DNaseI to avoid contatmination of genomic DNA using a PureLink DNase Kit (Invitrogen) according to the protocol. The concentration and purtity of the RNA were assessed using a NanoDrop Spectrophotometer (ND-1000, Thermo scientific). cDNA was synthezied from 1000 ng RNA using TaqMan reverse transcription Reagents (Life Technologies) and random hexamers to prime the reaction. Reverse transcription was performed at 25 °C for 10 min, 48 °C for 1 hour and 95 °C for 5 min. Reactions without TaqMan reverse transcriptase were used as negative controls in the real-time PCR study.
4
Total RNA Extraction and Quantification
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Standard techniques were used. Briefly, the total RNA extraction was undertaken using the PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA, USA). The PureLink™DNase kit (Invitrogen) was then used to remove genomic DNA. The integrity of RNA was assessed via agarose gel electrophoresis via the Experion automated electrophoresis station and the Experion StdSens Analysis kit according to the manufacturer’s instructions (Bio-Rad Laboratories Inc., Hercules, CA, USA), and total RNA was quantified using a spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific, Newark, DE, Waltham, MA, USA) with readings taken at 260 and 280 nm.
5
Transcriptome Profiling of VRC Treatment
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Total RNA extraction (hot phenol method), DNase treatment (PureLink DNase kit; Invitrogen Inc., Carlsbad, CA, USA) and quality checks of RNA samples by agarose gel electrophoresis were conducted as previously described [25 (link)]. Total RNA samples (3 each of VRC treated and untreated control samples) were submitted to Genewiz Biotechnology Co. Ltd. (now Azenta Life Science), Suzhou, China, for transcriptome sequencing. Sample quality control checks were performed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and a NanoDrop spectrophotometer (Thermo Fisher Scientific Inc. Waltham, MA, USA). RNA sequencing (RNA-seq) of 150 bp paired-end reads with a total of ~40 million reads was performed on NovaSeq 6000 (Illumina, San Diego, CA, USA).
6
RNA Extraction and RT-qPCR for Developmental Stages
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Total RNA was separately extracted from caryopses at different developmental stages, transgenic rice calli, and 5-day-old Arabidopsis seedlings using TRIzol reagent (Invitrogen, USA) and purified using a PureLink RNA Mini Kit (Invitrogen) and a PureLink DNase Kit (Invitrogen), according to the manufacturer’s protocol. Approximately 2 μg RNA was reverse transcribed using a Reverse-Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) and used as template for RT-PCR and RT-qPCR. PCR reactions were carried out in a MyCycler thermal cycler (Bio-Rad, USA) using extension cycling conditions of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The amplifications were performed for 28–33 cycles. qRT-PCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, USA) with Power SYBR Green PCR Master Mix (Applied Biosystems). The thermal program was 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. The dissociation curve program was used to confirm the specificity of the target amplification product. Three independent biological replicates were performed for RT-qPCR analyses. OsActin1 or AtUBQ10 was used as an internal control in both qRT-PCR and RT-PCR analyses. The primers for the target genes are listed in S4 Table .
7
Expression Analysis of OsSUB Genes
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Here, we focused on OsSUB29, OsSUB53, OsSUB58 and OsSUB63, while OsASP1 was used as a control (Chang et al., 2020 (link)). Total RNA from caryopses at different developmental stages was extracted using TRIzol® reagent according to the manufacturer’s protocol (Invitrogen, United States). Combined with the PureLink® DNase kit (Invitrogen), we used the PureLink® RNA Mini Kit (Invitrogen) to purify total RNA. To generate cDNA, approximately 2 μg of total RNA was reverse-transcribed utilizing the Reverse-Aid™ First Strand cDNA Synthesis Kit (Fermentas, Canada). Using PCR Master Mix (Power SYBR® Green; Applied Biosystems, United States), we conducted qRT–PCR on a 7,500 Fast qRT–PCR System (Applied Biosystems). The thermal cycle was 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. We examined the target amplification product specificity using a dissociation curve program. We performed three independent biological replicates of qRT–PCR, and OsActin3 was used as an internal control. |A fold change in the relative gene expression value| ≥ 2 indicates a significant difference between any two samples. The primers are shown in Supplementary Table 3 .
8
RNA Extraction from Frozen Tissue
9
Extraction and Quality Control of RNA from Mouse Kidneys
10
Comprehensive Transcriptome Analysis by Iso-Seq
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Total RNA was extracted using the a PureLink RNA Mini Kit (Invitrogen Inc., Carlsbad, CA, USA), followed by DNase digestion and RNA puri cation using an on-column PureLink DNase Kit (Invitrogen Inc.) according to the manufacturer's instructions. 1% agarose gel was used to monitor whether there existed RNA degradation and potential contamination. The purity of RNA samples was determined by using a NanoPhotometer Spectrophotometer (Implen, Westlake Village, CA, USA). RNA concentration was measured using a Qubit 2.0 Fluorometer (Invitrogen Inc.). RNA integrity was checked using an RNA Nano 6000 Assay Kit on a BioAnalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) before sequencing library preparation.
Construction of Iso-Seq complementary DNA (cDNA) library and PacBio sequencing Construction of Iso-Seq cDNA library and PacBio Sequencing were performed at Novogene Co., Ltd (Beijing, China). The mRNA was enriched using oligo-dT magnetic beads from 4.0 μg total RNA and reverse transcribed into cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, now Takara, http://www.takarabio.com). The size-selected cDNA library was constructed according to the BluePippin Size Selection System protocol as described by PacBio (PN 100-092-800-03) and sequenced on the PacBio Sequel platform.
Construction of Iso-Seq complementary DNA (cDNA) library and PacBio sequencing Construction of Iso-Seq cDNA library and PacBio Sequencing were performed at Novogene Co., Ltd (Beijing, China). The mRNA was enriched using oligo-dT magnetic beads from 4.0 μg total RNA and reverse transcribed into cDNA using the SMARTer PCR cDNA Synthesis Kit (Clontech, now Takara, http://www.takarabio.com). The size-selected cDNA library was constructed according to the BluePippin Size Selection System protocol as described by PacBio (PN 100-092-800-03) and sequenced on the PacBio Sequel platform.
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