Trelief animal genomic dna kit

The sgRNAs targeting exon24 of Bag6 in the mouse genome were designed according to the design principle and program of CRISPR/Cas9 (http://crispr.mit.edu/). The T7‐sgRNA plasmid was transcribed in vitro with MEGAshortscript™ T7 Transcription Kit (AM1354, Invitrogen) and the RNA was purified with MEGAclear™ Transcription Clean‐Up Kit (AM1908, Invitrogen). Cas9‐encoding mRNA (50 ng/μl, A29378, Thermo Fisher) and sgRNAs (50 ng/μl) were co‐injected into one‐cell‐stage wild‐type BDF1 embryos. The embryos were cultured in 5% CO₂, 37°C incubators for 24 h and put into the oviduct of pseudo‐pregnant BDF1 female mice. Total genomic DNAs of F0 mice were extracted according to Trelief™ Animal Genomic DNA Kit (TSP201‐50, Tsingke).

All 160 POC samples were processed using the KaryoLite™ BoBs™ assay kit (PerkinElmer, Turku, Finland) according to the manufacturer’s protocol. First, genomic DNA was extracted using the Trelief™ Animal Genomic DNA Kit (TSINGKE Biological Technology Inc., Beijing, China). Second, the extracted DNA was labeled with biotin, purified using a purification kit, and incubated overnight with the BoBs™ probes (fluorescently coded Luminex® beads with DNA probes). Thereafter, the hybridized beads were washed and bound to the reporter molecule (streptavidin phycoerythrin), and washed again. The DNA signals on the microbeads were measured using the Luminex® 200™ instrument system, and the experimental results were analyzed using the BoBsoft™ software. The detailed BoBs workflow is described elsewhere [24 (link), 25 (link)].

Cells were collected from the shNC + NC group, shSPESP1 + NC group, shNC + siMeCP2 group and shSPESP1 + siMeCP2 group for MSP detection. First, cell DNA fragments (TreliefTM Animal Genomic DNA Kit, TsingKe) were extracted, and DNA samples were converted into bisulfite using a DNA bisulfite conversion kit (DP215‐02, TIANGEN). Further purification was performed using the methylation‐specific PCR kit (EM101‐01, TIANGEN). PCR products were analysed with 3% agarose gel and quantified with ImageJ software.

Part epidemic tissues of the fifth-instar larval mutants of four genes (white, brown, ok, lightoid) or the thorax and abdomen of adults (white, scarlet) and their corresponding wild types were dissected in phosphate buffer saline and then used to extract genomic DNA using TreliefTMAnimal Genomic DNA Kit (TsingKe, China) following the manufacturer’s protocols. The tissues of each individual were as a biological sample. At least three replicates were carried out for mutants of each gene. Except some adult mutants of white gene, part tissues of the same individual for the mutants and wild-types are also used for RNA extraction as described in the following part. Subsequently, primers flanking the target sites for each gene (Additional file 1: Table S4) were designed, and the PCR reaction were carried out using the 20 μl volumes, according to TransDirect PCR SuperMix (Trans, China). PCR products were TA-cloned into PMD19 vectors (Takara, Japan) and 10 clones were randomly picked up and sequenced for each individual. Sequence data were analyzed using SeqMan software (DNASTAR7.0) to determine the exact mutation type.