Thermal cycler

The PCR of 16S rRNA gene from isolated DNA was amplified using a universal oligonucleotide primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) (Lane, 1991 ) in a thermal cycler (Thermo Fisher, United States). The reaction mixture, conditions, and protocol for the polymerase chain reaction amplification were done following the method of Chagnaud et al. (2001) (link). The PCR amplification was performed in a mixture containing a final volume of 50 μl of GoTaq Green Master Mix (2×) (M7122, Promega, United States), 10 μM of F primer, 10 μM of R primer, and nuclease-free water (NEB). The PCR reaction program was set under the following PCR conditions: 94°C for 10 min; 94°C for 1 min, 65°C for 1 min, and 72°C for 30 s for 35 cycles; and 72°C for 7 min. The PCR products were detected by electrophoresis using 1% agarose, and the bands were stained with 7 μl/100 ml of ethidium bromide and visualized using Gel Doc EZ Imager (Bio-Rad, United States). A standard 100-base pair DNA ladder was used for the verification of amplicon size. The amplified PCR products were purified using the PEG (polyethylene glycol)–NaCl (sodium chloride) (20% w/v of PEG, 2.5 M NaCl) precipitation method of Schmitz and Riesner (2006) (link).

After adjusting the RNA concentration of all isolated RNA samples, synthesis of cDNA was done using revertAid First Strand cDNA Synthesis Kit (Thermofisher, USA). The reaction mix consisted of 1 μl of oligo (dT) 18 primer, 11 μl of the adjusted RNA, 4 μl of 5 × reaction buffer, and 2 μl of 10 mM dNTP for each RNA sample. Finally, 1 μl of RiboLock RNase inhibitor and 1 μl of revertAid reverse transcriptase were added to reach a final volume of 20 μl. The samples were incubated in a thermal cycler (Thermofisher Scientific, CA, USA) for 60 min at 42 °C followed by 70 °C for 5 min. The cDNA samples were stored in − 20 °C freezer till used for real-time PCR runs.

Maxima First Strand complementary DNA (cDNA) synthesis kit (Thermo Fisher Scientific Inc., San Diego, CA, USA) was used to convert the total RNA (1 µg) into cDNA, which were then loaded into a thermal cycler (Labnet, USA). The cDNA was then used to quantify the selected gene expressions (Table 1) on the Eco^TM Real-Time PCR System (Illumina Inc., San Diego, CA, USA) using a Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific Inc., San Diego, CA, USA). The mixture was prepared by mixing 12.5 µL of Maxima SYBR Green qPCR Master Mix, 0.3 µM of forward and reverse primers and ≤ 500 ng of cDNA. Nuclease-free water was used to top up the mixture up to 25 µL. The mixture was then run using the Eco^TM Real-Time PCR System (Illumina Inc., San Diego, CA, USA) platform and the reaction was set at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15s, and annealing/extension at 55–60 °C for 15 to 30s.

Total RNA was extracted from frozen hippocampal tissue using Eastep® Super Total RNA Extraction Kit (Promega, China) according to the manufacturer's instructions. cDNA synthesis and qRT‐PCR were performed with HiScript Q RT SuperMix for qPCR (Vazyme, China) according to the kit instructions. Relative mRNA expression levels were determined with KAPA SYBR® FAST qPCR Kits (KK4601, Roche) by using a thermal cycler (Thermo Fisher Scientific Inc.). All samples were run in triplicate, and the glyceraldehyde phosphate dehydrogenase (GAPDH) gene served as the internal reference.

Reverse transcription was conducted with the PrimeScript RT reagent Kit with gDNA Eraser for RT-PCR (RR047A, Takara Bio Inc., Beijing, China) and the thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA) in a volume of 20 µL. The single cycle was 35 °C for 15 min and the incubation period was 85 °C for 5s. Relative qPCR was done with the Bio-Rad CFX Opus 96 Instrument and TB Green® Premix (RR820A, Takara Bio Inc., Beijing, China). Fold induction was calculated using the comparative Ct method and the formula 2^-(ΔΔCt).

For saving cost and time, the crRNAs used in this study were prepared by in vitro transcription (IVT). The ssDNA template with the reverse complementary sequence of the crRNA was synthesized with a T7 promoter (Sangon, China). An annealing reaction mix consisted of 1 µL of DNA template (100 µM), 1 µL of Standard Taq buffer (10×), 1 µL of T7-3G-IVT primer (100 µM, Supplementary Table 6), and 7 µL of DNase/RNase-free water. The master mix was denatured at 95 °C for 5 min, followed by gradient annealing with the ramp rate at 0.1 °C/s until cooling to 4 °C using a thermal cycler (Thermo, USA). Then, the annealing products were transcribed at 37 °C for 8 ~ 12 h via HiScribe T7 High Yield RNA Synthesis Kit according to the manufacturer’s instructions. The transcribed crRNA with remaining DNA was treated with DNase I, followed by RNA purification via RNA Clean & Concentrator-5 Kit. Finally, the crRNA concentration was quantified by the Qubit™ Flex Fluorometer (Thermo, USA) via the Qubit™ RNA Broad Range Assay Kits, and stored at −80 °C until use.