Longamp buffer

Genomic DNA was isolated from each well of a confluent 24- or 96-well plate as follows: cells were incubated for 2 h at 55°C in 20 or 40 μl of lysis buffer (0.45% NP40, 0.45% Tween20, 1× NEB LongAmp PCR buffer) and subsequently heated to 95°C (10 min). One to two microlitres of this lysate was used in a 10 μl PCR reaction. PCR reactions comprised 0.2 μl DMSO (100% v/v, Sigma), 0.3 μl dNTPs (10 mM, Thermo Fisher Scientific), 2.0 μl LongAMP buffer (5× NEB), 0.4 μl LongAMP Taq (NEB) and 12 pmol of each primer. Thermal cycling was performed using the following conditions: 1 cycle 94°C for 3 min; 40 cycles 94°C for 15 s, 60°C for 30 s, 65°C for 2 min; followed by final extension at 65°C for 10 min.
For each targeted locus, two sets of genotyping primers spanning the junction of genomic sequences and targeting vector were used (left and right arms). Gene-specific primers were designed outside the 5′ and 3′ homology arms and were used in combination with primers in the knock-in cassette (either CAG-LUC-2A-GFP-IRES-BSD for targeting Rosa26 and AAVS1 loci, or Ef1α-Puromycin for the knockout experiments). To identify NHEJ-based indel formation on the second, non-targeted alleles, the region flanking the sgRNA target sites (500-600 bp) was amplified using PCR with gene-specific primers and directly assessed by Sanger sequencing. Sequences of all primers are provided (Table S2).

To study the function of differentially expressed miRNAs identified from hypoxia at rhizosphere, a popular technique, short tandem target mimic (STTM), was employed to block miR171 and miR390’s expression in Arabidopsis. STTM vectors construct were generated by the following procedures described by Tang et al. [13 (link)]. In brief, specific STTM primers were designed and synthesized. PCR was carried out by using LongAmp, dNTPs, and LongAmp buffer from New England BioLabsInc (NEB). STTM fragment was inserted into the pOT2-Poly-Cis vector firstly by PCR, then through SwaI digestion (NEB), and lastly via T4 DNA ligase (NEB) catalyzed ligation. The STTM containing pOT2-STTM vector was amplified in DH5α competent cells. We removed the replication origin from pOT2-STTM by origin deletion PCR. The origin deleted pOT2-STTM together with pFGC5941-PacI binary vector was incubated with PacI endonuclease (NEB) and then were ligated together by T4 DNA ligase (NEB). The STTM-pFGC5941 plasmid was amplified in DH5α competent cells. All the prepared STTM-pFGC5941 plasmids for different miRNAs were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ones with exactly correct sequence were used to transform the plants. Transgenic Arabidopsis lines were selected by spraying 0.1% glufosinate (bar herbicide).