Rneasy minelute

To construct TALEN-L and TALEN-R plasmids, TAL repeats were assembled by the modified Golden Gate assembly method (Sakuma et al, 2013 (link)). TALEN plasmids and the phiC31 integrase expression plasmid (Ishikawa et al, 2018 (link)) were linearized with NotI, purified by phenol/chloroform extraction, and used as a template to synthesize capped mRNAs using the mMESSAGE mMACHINE SP6 kit (Life Technologies) followed by purification with RNeasy MinElute (QIAGEN). Synthesized RNAs were microinjected as described previously in Ishikawa et al (2011) (link) into one-cell-stage embryos at the concentration of 50 ng/μl for TALEN-L and TALEN-R and 100 ng/μl for the phiC31 integrase expression plasmid.

Total RNA was extracted from leaf, stem, silk, husk, ear and pollen tissues using Trizol and RNeasy MinElute (Qiagen) RNA clean up kit, with an Dnase I treatment of 20 min^{69 (link)}. The RNA integrity was assessed with a Bioanalyzer prior to the construction of the Iso-Seq library. The Iso-Seq libraries were prepared and sequenced by Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida using a PacBio Sequel system. PacBio Iso-Seq data were analyzed by running the IsoSeq3 v3.1 in PacBio SMART Analysis v7.0 (https://github.com/PacificBiosciences/IsoSeq3) to generate high-quality, full-length transcript sequences.

To construct TALEN, TAL repeats were assembled by the modified Golden Gate assembly method (Sakuma et al., 2013 (link)).
To construct sgRNA vectors for CRISPR-Cas9, two oligonucleotides corresponding to the 5’-side of exon 4 of the XBP1 gene (5'-taggCTGCGGACTCAGAAGACC-3' and 5'-aaacGGTCTTCTGAGTCCGCAG-3'), and two oligonucleotides corresponding to the 3’-side of exon 4 of the XBP1 gene (5'-taggTGCCTCCGCAGCAGGTGC-3' and 5'-aaacGCACCTGCTGCGGAGGCA-3') were annealed and ligated into BsaI-digested DR274 vector (Addgene). sgRNA vectors were linearized with DraI, purified by phenol chloroform extraction, and used as template to synthesize sgRNAs using T7 RNA polymerase.
TALEN and Cas9(D10A) expressing vectors were linearized with NotI, purified by phenol chloroform extraction, and used as template to synthesize capped mRNAs using the Message mMachine SP6Kit (Life Technologies, Gaithersburg, MD).
Synthesized RNAs were purified by RNeasy MinElute (Qiagen, Germany) and microinjected into one-cell stage embryos at the concentration of 50 ng/μl for both left and right TALEN, 100 ng/μl for Cas9(D10A), and 25 ng/μl for both sense and antisense strand sgRNA of Cas9. Injection was performed as described previously (Ishikawa et al., 2011 (link)).

After homogenization in liquid nitrogen using mortar and pestle, total RNA was extracted using a CTAB based extraction method, modified after [81 (link)]. After precipitation and resuspending the RNA, a DNase I digestion was performed, using Qiagens RNase-Free DNase Set (Cat. no. 79254). Afterwards, the DNase was removed using silica columns (Qiagen RNEasy MinElute, Cat. no. 74204). The integrity of the total RNA was checked on a 2100 Bio-analyzer (Agilent, CA, USA) using the RNA 6000 nano assay and the Plant total RNA protocol. The purity of total RNA was checked on a Nanodrop ND-1000 (Thermo Scientific, Bremen, Germany). From each sample, one deep mRNA sequencing library was prepared, using the TruSeq RNA Sample Preparation Kit v2 starting from 4 μg total RNA (Illumina, CA, USA). The libraries were prepared and sequenced on two Illumina 100 bp paired end (PE) flow cells on the Illumina HISEQ 2000 at the Genome Quebec Innovation Centre in Montreal, Canada. Based on earlier investigations [82 (link)], we aimed at an effective sequencing depth of 20 million aligned reads per sample.

Individual embryos were lysed in 300 µl Trizol with a 5 mm stainless steel bead (Qiagen) for 2 min at 20 Hz in a tissue lyser (Qiagen). After mixing of 200 µl chloroform to the homogenate and incubation for 30 min at room temperature the upper phase was transferred to a new tube. One volume of fresh 70% ethanol was added and the RNA was purified over a spin column (Qiagen RNeasy MinElute). After quantification (Qubit RNA BR) the sample was treated with DNAse enzyme (Qiagen) and purified over a spin column again. Up to 500 ng total RNA was used for the generation of strand-specific RNA-seq libraries containing unique index sequences in the adapter. Libraries were pooled and sequenced on Illumina HiSeq.