Kapa hifi hotstart pcr mix

Wild type (WT) SpCas9 was complexed with guide RNAs targeting introns 36 and 38 (see Supplementary Data 5) along with PAM killing ssDNA templates (Supplementary Data 5) and then microinjected into the cytoplasm of C57BL/6JArc zygotes. Injected zygotes were then transferred to the oviduct of pseudo-pregnant females for in-utero development. The two single-stranded oligos were utilized to insert PAM killing mutations at the intronic cut sites to reduce the proportion of alleles that may be repaired by Non-Homologous End Joining (NHEJ) between the two cut sites which would delete exons 37 and 38 (see Fig. 1a). After successful pregnancy and birth, pups were genotyped using a rapid PCR strategy (Supplementary Fig. 1j) and deletion of Exon 37 and 38 was confirmed by Sanger sequencing. For genotyping the founder animals, genomic DNA (gDNA) was isolated from a small ear notch (also served as an animal identifier) using High Pure PCR Template Preparation Kit (Roche, 11796828001) as per manufacturer’s instructions. PCR was performed with KAPA HiFi HotStart PCR mix (Roche, KK2601) (details in Supplementary Data 5).

Pancreatic tissue DNA was enriched for 16S rRNA in a preamplification PCR using primers 331F (5’-TCCTACGGGAGGCAGCAGT-3’)61 (link) and 979R (5’-GGTTCTKCGCGTTGCWTC-3’).62 (link) The cycling conditions consisted of an initial template denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 65°C for 20 s, extension at 72°C for 30 s and a final extension at 72°C for 10 min. This was followed by a size-selective cleanup using SPRIselect magnetic beads (0.8 left-sized; Beckman Coulter, Brea, California, USA). Faecal and salivary DNA were not preamplified.
Targeted amplification of the 16S rRNA V4 region (primer sequences F515 5’-GTGCCAGCMGCCGCGGTAA-3’ and R806 5’-GGACTACHVGGGTWTCTAAT-3’),63 (link) was performed using the KAPA HiFi HotStart PCR mix (Roche, Basel, Switzerland) in a two-step barcoded PCR protocol (NEXTflex 16S V4 Amplicon-Seq Kit; Bioo Scientific, Austin, Texas, USA) with minor modifications from the manufacturer’s instructions. PCR products were pooled, purified using size-selective SPRIselect magnetic beads (0.8 left-sized) and then sequenced at 2×250 bp on an Illumina MiSeq (Illumina, San Diego, California, USA) at the Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg.

gDNA from cells was extracted using DNeasy Blood and Tissue Kit (Qiagen), according to the manufacturer’s protocol. Next generation sequencing libraries were prepared as follows. Briefly, 4–10 μg of input gDNA was amplified by PCR with primers that amplify 150 bp surrounding the sites of interest (Supplementary Table 1b) using KAPA Hifi HotStart PCR Mix (Kapa Biosystems). PCR products were gel purified (Qiagen Gel Extraction kit), and further purified (Qiagen PCR Purification Kit) to eliminate byproducts. Library construction was done with NEBNext Multiplex Oligos for Illumina kit (NEB). 10–25 ng of input DNA was amplified with indexing primers. Samples were then purified and quantified using a qPCR library quantification kit (Kapa Biosystems, KK4824). Samples were then pooled and loaded on an Illumina Miseq (150 bp paired-end run or 150 single-end run) at 4 nM concentrations. Data analysis was performed using CRISPR Genome Analyzer^{53 (link)}.