The experimental design and cell harvesting for RNAseq analysis were performed as described for the RT-qPCR analysis. Three biological replicates from each time point of control and induced cultures were ground to a powder in liquid N2. Total RNA was extracted using ReliaPrep RNA Miniprep System (Promega) followed by DNAse treatment (DNAse RQ1, Promega), according to manufacturer’s instructions. RNA integrity/quality and concentration were assessed using Agilent Bioanalyzer RNA 6000 pico kit in a Bioanalyzer 2100 (Agilent Technologies) and Qubit 2.0 (Thermo Fisher Scientific), respectively. RIN (RNA integrity number) scores of above 7 were used. Poly-A mRNA isolation and cDNA libraries construction were carried out using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and NEBNext Ultra II Directional RNA Library Prep kit for Illumina (New England Biolabs), respectively, according to manufacturer’s protocols and using unique barcode for each sample. Pooled samples were sequenced in a high-output paired-end run (2 × 150 bp) using an Illumina® NextSeq 500.
Reliaprep rna miniprep system
Manufactured by Promega
Sourced in United States, Germany, Japan
The ReliaPrep™ RNA Miniprep Systems are a set of products designed for the extraction and purification of RNA from a variety of sample types. The systems utilize spin column technology to efficiently isolate high-quality RNA that can be used in downstream applications such as RT-qPCR, RNA sequencing, and other RNA-based analyses.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (10 mentions)
Goscript reverse transcription system, by Promega (10 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (8 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Lightcycler 96, by Roche (6 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (6 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Ssofast evagreen supermix, by Bio-Rad (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Gotaq qpcr master mix, by Promega (5 mentions)
Accupower 2x greenstar qpcr master mix, by Bioneer (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit, by Takara Bio (4 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (4 mentions)
Sybr green, by Promega (4 mentions)
Taqman probe, by Thermo Fisher Scientific (4 mentions)
151 protocols using reliaprep rna miniprep system
1
RNA-seq analysis of induced cultures
2
RNA Extraction from Thawed Tissue
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Tissue samples were thawed on ice, homogenized using a pistil and then extracted with the ReliaPrep RNA Miniprep system (Promega, Germany) for tissues as according to the manufacturer’s instructions. The RNA was quality checked; only samples with A260/280 ~ 2.0 and A260/230 ~ 2.0–2.2 were used. The RNA integrity index was determined with the help of an Experion microchip reader (Biorad, Germany) and a StdSens RNA kit. Only samples with a RIN > 8.0 were taken for qPCR. RNA quantity was determined with a Qubit RNA broad range assay kit (Thermo Fisher Scientific, Germany).
3
RNA Extraction and qRT-PCR Analysis
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Right lung tissues were lysed for RNA extraction using the ReliaPrep RNA Miniprep System (Promega Corporation, Madison, WI) as per the manufacturer's protocol. The total RNA concentration and purity was measured by spectrophotometry using a Nanodrop ND‐1000 system (Thermo Fisher Scientific, Waltham, MA). Approximate A260/A230 and A260/A280 ratios of two were considered ideal for RNA purity.63 First‐strand cDNA was synthesized from 1 μg purified RNA using a high‐capacity cDNA reverse transcription kit (Thermo Fisher Scientific). The relative levels of mRNA were measured by SYBR Green detection (Promega) in a PCR Mastercycler Ep Realplex system (Eppendorf, Hamburg, Germany). All samples were measured in triplicate. The relative TGF‐β1 transcript level was calculated as the ratio of the levels of the target gene (i.e., TGF‐β1) over the control gene (ie, acidic ribosomal phosphoprotein P0, 36B4). The primer sequences used in this study were: forward CAACCCAGCTCTGGAGAAAC and reverse GTTCTGAGCTGGCACAGTGA for 36B4; forward CTAATGGTGGACCGCAACAAC and reverse GACAGCCACTCAGGCGTATC for TGF‐β1.
4
Quantitative RT-PCR for Gene Expression
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RNA from whole cell lysates was isolated using the ReliaPrep™ RNA Miniprep System (Promega, Madison, WI, USA), according to the manufacturers’ protocol. For mRNA reverse transcription, 1μg of RNA was transcribed using the following reagents: 5× RT buffer and M-MLV RT 50.000 U (Promega), dNTP Mix (Promega), random hexamer primer (ThermoFisher Scientific), and RiboLock (Thermo Fisher Scientific). Reverse transcription was carried out at 25 °C for 5 min, 40 °C for 60 min, and 70 °C for 10 min. Sybr green-based real-time PCR (Maxima SYBR Green/ROX qPCR Master Mix, Thermo Fisher Scientific) was performed with the following protocol: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C, followed by 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. Individual samples were run in triplicates. The following primers were used: hNPNT fw: GGAGGCAAACACAGATCACC, hNPNT rev: TCCAATCTCCCCAGTGTGAC, hHPRT fw: GACCAGTCAACAGGGGACAT, and hHPRT rev: AACACTTCGTGGGGTCCTTTTC. Data was analyzed by using the ΔΔCt method.
5
Quantitative RT-PCR Analysis of Oxidative Stress Genes
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Total RNA was extracted from cells using a ReliaPrep RNA Miniprep System (Promega) according to the manufacturer's protocol. Purified RNA was subjected to reverse transcription with PrimeScript Reverse Transcriptase (Takara Bio, Otsu, Japan) and random hexamers (Takara). Quantitative real-time RT-PCR was performed using a CFX Connect™ RT-PCR System (Bio-Rad, Hercules, CA, USA) with SYBR® Premix ExTaq™II (Takara, RR820B), as previously described [20] (link), [21] (link). Transcripts of p62, Ho-1, Nqo1 and ribosomal protein S18 (Rps18) were amplified and Rps18 was used for normalization. Primer sequences are shown in Table 1 .Table 1
Primer sequence.
| Ho-1 | Forward | 5'-GAACTTTCAGAAGGGTCAGGTG-3' |
| Reverse | 5'-AGGGAAGTAGAGTGGGGCATAG-3' | |
| Nqo-1 | Forward | 5'-CGAATCTGACCTCTATGCTATGAAC-3' |
| Reverse | 5'-GAACTGAAATATCACCAGGTCTGC-3' | |
| p62 | Forward | 5'-TGGTGGGAACTCGCTATAAGTG-3' |
| Reverse | 5'-CCAAAGTGTCCATGTTTCAGC-3' | |
| MnSOD | Forward | 5'-CCCAAAGGAGAGTTGCTGGAG-3' |
| Reverse | 5'-CGACCTTGCTCCTTATTGAAGC-3' | |
| Gpx4 | Forward | 5'-TCTGTGTAAATGGGGACGATG-3' |
| Reverse | 5'-AGGGGCACACACTTGTAGGG-3' | |
| Rps18 | Forward | 5'-TGCGAGTACTCAACACCAACAT-3' |
| Reverse | 5'-CTTTCCTCAACACCACATGAGC-3' |
6
RNA-Seq of e14.5 CD133+ aNPC
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Total RNA from FACS purified e14.5 CD133-positive aNPC was extracted using Reliaprep RNA miniprep system (Promega, Leiden, Netherlands). Two brains of each genotype were pooled to get one biological replicate. The sequencing was performed by Beckman Coulter Genomics. Total RNA was further purified by ribodepletion and cDNA synthesis and library construction was performed on an automated Illumina TruSEQ stranded RNA-Seq library construction with a Biomek liquid handling platform. Detection of sequencing fragments was performed using the Illumina HiSeq 2500 platform. Alignment was made with Bowtie/Tophat, gene counts with Cufflinks and expression values/differential expression with edgeR. The sequencing output was on average 70.106 reads per sample.
7
RNA-seq of Genetic Knockouts
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RNA-seq was performed in triplicates per condition, including three independently derived clonal lines per genetic knock-out, and two RGd2 lines as wild-type controls. Exception is RGd2-N16h samples for which only 2 replicates were sequenced, as one was lost during library preparation. RGd2-2 is a clonal line derived from the parental RGd2 line (RGd2-1). Total RNA was extracted with ReliaPrep RNA Miniprep System (Promega) and was processed with Ribo-Zero capture probes (Illumina). Libraries were produced using NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific). Libraries were sequenced in the Illumina platform in paired-end mode.
8
Auxin Inducibility of GLV Genes in Arabidopsis
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To compare auxin inducibility of GLV6 and GLV10 expression between Col‐0 and gLBD16‐SRDX or lbd16lbd18lbd33 lines, 7‐d old seedlings grown on nylon membranes were transferred to medium containing 10 µM 1‐naphthalene acetic acid (NAA; Duchefa Biochemie) or an equivalent volume of dimethylsulfoxide (DMSO). Whole roots were sampled at the indicated time points after NAA or mock treatment. Roots of estradiol inducible iGLV6, rgi1rgi4rgi5/iGLV6 and mpk6‐3/iGLV6 seedlings, were sampled 12 d after germination (DAG) on solid ½MS medium supplemented with 2 µM estradiol (Sigma‐Aldrich) or DMSO. Peptide treated roots were sampled from 7 DAG seedlings that were incubated for 24 h in liquid ½MS, supplemented with 1 µM GLV10p or TOLS2p. Total RNA was isolated using the ReliaPrep™ RNA Miniprep System (Promega, Madison, WI, USA). Complementary DNA (cDNA) was synthesized with the qScript® cDNA SuperMix (Quantabio, Beverly, MA, USA). Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) experiments were performed using SYBR® Green Mix (Roche, Basel, Switzerland) in a LightCycler® 480 (Roche). CKA2 and CDKA1 were included as housekeeping genes (all primer sequences; Supporting Information Table S1 ).
9
Comprehensive Small RNA Sequencing Protocol
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In brief, total RNAs were extracted using the ReliaPrep RNA Miniprep System (Promega, Madison, WI, USA), according to the manufacturer’s protocol. Qualified RNAs at 8 μg with a high integrity number (RIN > 8.0) were subjected to library construction using the NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB, Ipswich, MA, USA), according to the manufacturer’s instructions, and sequenced on an Illumina Hi-Seq 4000 platform. Preliminary reads were trimmed, filtered, and aligned to the mouse reference genome (GRCm37), and small RNA high-quality reads were extracted and analyzed using the CLC Genomic Workbench (CLC bio, Aarhus, Denmark).
10
Quantitative RT-PCR Analysis of SMG Transcripts
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Total RNA was extracted from the SMG using a ReliaPrep RNA Miniprep System (Promega, Madison, WI) and reverse-transcribed to cDNA using a High-Capacity cDNA RT Kit (TOYOBO). The resulting cDNA was subjected to quantitative RT-PCR analysis using the KOD SYR qPCR Mix (TOYOBO, Osaka, Japan) in a Takara PCR Thermal Cycler Dice Gradient instrument (Takara Bio, Shiga, Japan). The specific primers used were as follows: EGF F:5ʹ-TGGAACCCAGTGGAATCAC-3ʹ; R:5ʹ-TGGGATAGCCCAATCCGAGA-3ʹ; NGF F:5ʹ-TTTTGATCGGCGTACAGGCA-3ʹ; R:5ʹ-CTGTCACTCGGGCAGCTATT-3ʹ; K18 F:5ʹ-TCAAGATCATCGAAGACCTGAGG-3ʹ; R:5ʹ-GCGCATGGCTAGTTCTGTC-3ʹ; K19 F:5ʹ-GGGGGTTCAGTACGCATTGG-3ʹ; R:5ʹ-GAGGACGAGGTCACGAAGC-3ʹ; Pmepa 1 F:5ʹ-TGGAGTTCGTGCAAATCGTG-3ʹ; R:5ʹ-TCCGAGGACAGTCCATCGTC-3ʹ; Crisp 3 F:5ʹ-ACAGTGGCCATTATCCAAGCA-3ʹ; R:5ʹ-GCATGTAGCTAGGCAACGTTTT-3ʹ; KLK 1 F: 5ʹ-CACCCGTCAAATATGAATACCCA-3ʹ; R: 5ʹ-TAGGGCCCCATGATGTGATAC-3ʹ; Cftr F: 5ʹ-CTGGACCACACCAATTTTGAGG-3ʹ; R: 5ʹ-GCGTGGATAAGCTGGGGAT-3ʹ; Runx 1 F: 5ʹ-CTGCAACAAGACCCTGCCCATCGCTTTC-3’; R: 5’-CTCCGCCCGACAAACCTGAGGTCGT-3’; p130Cas F: 5’-CCAAAGCCCTCTATGACAATGT-3’; 5’-CTTGAGGCGGTTACCAGGC-3’; GAPDH F:5ʹ-AGGTCGGTGTGAACGGATTTG-3ʹ; R:5ʹ-TGTAGACCATGTAGTTGAGGTCA-3ʹ; Androgen Receptor F:5ʹ-CTGGGAAGGGTCTACCCAC-3ʹ; R:5ʹ-GGTGCTATGTTAGCGGCCTC-3ʹ.
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