Rnaqueous rna isolation kit

Total RNA was extracted from seedlings and different organs of mature plants using an RNAqueous RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA), supplemented with Plant RNA Isolation Aid (Life Technologies, Carlsbad, CA, USA). First-strand complementary DNA (cDNA) synthesis, semi-quantitative RT-PCR, and quantitative RT-PCR were performed following the procedures described by Seok et al. [50 (link)]. The primers used for PCR are listed in Supplementary Table S1.

Total RNA was extracted using an RNAqueous RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA), supplemented with Plant RNA Isolation Aid (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ instructions. Two micrograms of total RNA was reverse-transcribed in a total volume of 25 μL containing 0.5 μg of oligo dT primer, 0.5 mM dNTPs, and 200 units of Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Madison, WI, USA).

Total RNA extraction was conducted using an RNAqueous RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) and a Plant RNA Isolation Aid (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Subsequently, 2 μg of total RNA was reverse-transcribed in a total volume of 25 μL containing 0.5 μg of oligo dT primer, 0.5 mM dNTPs, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI, USA).

RNAqueous RNA isolation kit (Invitrogen, AM1931) was used for bulk RNA extraction as per the manufacturer protocol. Briefly, E14.5 cochlear ducts from both inner ears of one embryo were microdissected in DEPC‐treated cold PBS, collected in RNA extraction buffer and homogenized (Bel‐Art 650 000 000). RNA extraction and subsequent removal of genomic DNA were performed according to the manufacturer's protocol. Three littermates per genotype were used. The yield and integrity of total RNA from microdissected samples were measured using a 2100 Bioanalyzer (Agilent Technologies). Next generation sequencing and bioinformatics analysis were performed at Northwestern Sequencing core. Briefly, TruSeq mRNA‐Seq Library Prep was used to create cDNA libraries according to manufacturer's protocol. Each library was sequenced to generate 50 base pair single reads on the Illumina HiSeq Sequencing (Illumina). The sequence reads were aligned to the mouse reference genome sequence (USCS mm10) using STAR aligner.⁵⁸ Alignments were assembled and annotated using Cufflinks.⁵⁹ DESeq2⁶⁰ was used to detect differentially expressed gene transcripts. The raw data from bulk RNA sequencing of E14.5 cochlear duct is deposited in Gene expression Omnibus (GSE193046) and can be accessed through the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193046

Total RNA was isolated using the RNAqueous RNA Isolation Kit (Invitrogen, Carlsbad, CA, USA) and Plant RNA Isolation Aid (Invitrogen, Carlsbad, CA, USA), in accordance with the manufacturer’s protocol. Total RNA (2 μg) was used for reverse transcription using Moloney murine leukemia virus reverse transcriptase (Promega Corp., Madison, WI, USA) as previously described [23 (link)].
RT-qPCR was conducted using a QuantStudio^TM 3 real-time PCR system (Applied Biosystems, Foster, CA, USA) and Power SYBR^TM Green PCR Master Mix (Applied Biosystems, Foster, CA, USA) in accordance with manufacturer’s manual. Real-time DNA amplification was analyzed using QuantStudio^TM Design and Analysis software (version 1.4.3) (Applied Biosystems, Foster, CA, USA). Three independent reactions were conducted for each technical replicate. Two technical replicates were conducted for each biological replicate.
Semi-quantitative RT-PCR was conducted in accordance with previous study [23 (link)]. PCR reactions were repeated 30–31 cycles for AtC3H12 and 23–24 cycles for GAPc.
Primers for RT-PCR are shown in Table S2.

For the samples collected from the time course of germination (Col-0 only; Fig. 1a(i)), the Ambion Plant RNA isolation aid and RNAqueous RNA isolation kit were used for RNA isolation. RNA quality and integrity were determined using the Nanodrop 1000 Spectrophotometer and Agilent Bioanalyser. Only high-quality RNA samples (Abs260/280 nm ratios of 2.0–2.1) were used for RNA-seq library generation with the Illumina TruSeq Total RNA sample prep kit. RNA-seq libraries were multiplexed and loaded per lane into the Illumina HiSeq flow cell v3. All sequencing protocols were carried out as per the manufacter’s instructions using the Illumina HiSeq 1000 and HiSeq control software.
For the samples collected from the eight TF mutant lines and Col-0 in parallel (validation of the DREM predictions), RNA from the 24 h SL timepoint was extracted with the Spectrum RNA extraction kit (Sigma) in duplicates for each genotype. RNA-seq libraries were prepared with the Illumina TruSeq mRNA kit, pooled and sequenced on one NextSeq500 flow cell.