Pcr purification column

Potential gRNA templates were generated by CHOPCHOP (http://chopchop.cbu.uib.no/). The chosen forward and reverse oligonucleotides, 20 bps upstream of the PAM sequence, were ordered with additional nucleotides added to the 5’ end to permit cloning into the pDR274 vector^{89 (link)}. The oligonucleotide forward sequence used was: 5’ - tag gAC TTT AGT GTC CAA AAG AA - 3’ and oligonucleotide reverse sequence used was: 5’- aaa cTT CTT TTG GAC ACT AAA GT – 3’. 2 uM of each oligonucleotide was mixed in annealing buffer (10 mM Tris, pH 8, 50 mM NaCl, 1 mM EDTA) and incubated at 90 °C for 5 min, then cooled to 25 °C over a 45 min time interval. The pDR274 vector was linearized with BsaI and oligonucleotides were ligated into the vector with T4 ligase (NEB) for 10 min at room temperature. The ligation reaction was transformed into competent cells and then plated on kanamycin LB plates. Selected colonies were grown, mini-prepped (Zyppy Plasmid Kits, Zymo Research), and Sanger sequenced. The gRNA DNA sequence was then PCR amplified from 50 ng μL⁻¹ of the plasmid with Phusion (NEB) and the following primers: F: 5’-GTTGGAACCTCTTACGTGCC-3’ and R: 5’-AAAAGCACCGACTCGGTG-3’. The PCR product was digested with DpnI at 37 °C for 1 h, heat inactivated at 80 °C for 20 min, and then purified with a Qiagen PCR Purification column. RNA was synthesized with a MEGAscript T7 Transcription Kit (Ambion).

Total RNA was prepared from the whole plant using Sigma Spectrum Plant Total RNA Isolation Kit and was further used for double stranded cDNA synthesis following the standard protocol. DS cDNA (5 μg) was purified using PCR purification column (Qiagen, GmbH, Germany). DNA was randomly sheared to an average fragment size 300–800 bp by applying 30 psi (2.1 bar) pressure of gaseous nitrogen for 1 minute using a nebulizer. The size of DNA fragments was analyzed on Agilent BioAnalyzer DNA 7500 Chip. Sheared cDNA ends were repaired by T4 DNA polymerase and T4 polynucleotide kinase. Small cDNA fragments were removed by Ampure beads purification. Sequencing library was prepared as per standard protocol (Roche, Switzerland) and the quality and quantity of the library was analysed on Agilent BioAnalyzer High sensitivity chip. An emulsion PCR based amplification of sequencing library was performed using GS emPCR kit and sequencing of the amplified fragments was done as per manufacture’s protocol on 454 Genome Sequencer FLX (Titanium) using half of the Pico Titer Plate (PTP, 70 × 75).

Genomic DNA was extracted from shrimp gut content using the PowerSoil DNA Isolation kit (MoBio Laboratories, Carlsbad, CA, USA) according to manufacturer’s instructions. The concentration and quality of the extracted DNA were assessed using a Nanodrop 2000 (NanoDrop Technologies, Wilmington, DE, USA) and agarose gel electrophoresis. The hypervariable V3-V4 region of the intestinal microbiota 16S rRNA genes were amplified using a set of barcoded fusion reverse primers and the same forward primer (Table S1). PCR amplification was carried out in a 50-μL reaction containing 1× EasyTaq PCR Supermix (Transgen Biotech, China), 0.2 μM of forward and reverse primers, and about 10 ng template DNA. The amplification conditions consisted of initial denaturation at 98 °C for 2 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s and final extension at 72 °C for 10 min. Amplicons were pooled and purified using a Qiagen PCR purification column. Finally, the PCR products was adjusted to the same concentration and submitted to the Genome Sequencing Company (Novogene, China) for paired-end library preparation, cluster generation, and 250-bp read sequencing on an Illumina HiSeq. 2500.

Plant genomic DNA was extracted with a modified CTAB method [40 (link)]. Taq polymerase (NEB) or Q5 High-Fidelity DNA polymerase (Thermo Scientific) were used for PCR amplification to detect deletion events. Annealing temperatures were tested and optimized for each pair of genotyping primers, with extension time kept short enough to favor amplification of fragments carrying deletions under 1 kb, but not the original, non-deleted fragment. T7E1 (T7 Endonuclease I, NEB) was used for identification of mutations at individual sgRNA target sites [41 (link)]. DNA fragments were amplified from genomic DNA using a pair of primers spanning the targets with Q5 High-Fidelity DNA polymerase (Thermo Scientific). The PCR product was purified using PCR Purification Column (Qiagen, Hilden, Germany) and concentration was determined with a Nanodrop 2000 Spectrophotometer (Thermo Scientific). 100 ng of purified PCR product was denatured-annealed under the following conditions: 95 °C for 2 min, ramp down to 85 °C, at 2 °C/s, followed by ramping down to 25 °C at 0.1 °C/s. Annealed PCR products were digested with 5 U of T7E1 for 20 min at 37 °C. The T7E1-digested products were separated on a 2% agarose gel. A similar amount of substrate DNA without T7E1 incubation was used as a negative control.