Axyprep magpcr clean up beads

RAG1A locus was PCR amplified by Fastpfu (Transgen Biotech, China) using listed primers (Supplementary Table S11). Amplicons recovered by 1.2× AxyPrep Mag PCR Clean-Up beads (Axygen, US) were cleaved by StyI (NEB) for an hour followed by agarose gel electrophoresis. Band intensity was quantified by Image J (version 1.51J8) and the indels was measured by the following formula: $$\mathrm{Indels}=\frac{I_{\mathrm{C}}}{I_{\mathrm{C}}+I_{\mathrm{U}}}$$ I_C is sum of the intensity of two cleaved bands, and I_U is the intensity of the uncleaved bands.

Up to 2 pieces of feces from individual mice were sterilely collected, heat inactivated and subjected to bead-beating using Lysing Matrix B, 0.1 mm silica spheres (MP Biomedicals). DNA was extracted using a column-based DNA stool mini kit (Qiagen) following the manufacturer’s protocol. Final DNA concentrations and quality were determined by fluorometric analysis (Qubit 2.0 Invitrogen) using quant-iT BR dsDNA reagent kits (Invitrogen) and normalized to a uniform concentration and volume. Microbial 16S rRNA amplicons were constructed by amplification of the V4 region of the 16S rRNA gene with universal primers previously developed against the V4 region, flanked by Illumina standard adapter sequences [19 (link), 20 (link)]. Dual-indexed forward and reverse primers were used in all reactions. Amplicon pools at equal concentrations were combined, thoroughly mixed, and purified using Axyprep MagPCR clean-up beads (Axygen) prior to final analysis on a fragment analyzer automated electrophoresis system (Advanced Analytical). Amplicon pools were then diluted following Illumina’s standard protocol for sequencing on a MiSeq instrument generating 2x250 bp paired-end reads.

Libraries were constructed by following the manufacturer’s protocol with reagents supplied in 10x Genomics Chromium Next GEM Single Cell 3′ Kit v3.1. Briefly, cell suspension concentration and viability were measured manually and with an Invitrogen Countess II automated cell counter. Cell suspension (900 cells per microliter), reverse transcription master mix, and partitioning oil were loaded on a Chromium Next GEM G chip with a cell capture target of 5000 cells per library. Post-Chromium controller GEMs were transferred to a PCR strip tube and reverse transcription performed on an Applied Biosystems Veriti thermal cycler at 53 °C for 45 min. cDNA was amplified for 12 cycles and purified using Axygen AxyPrep MagPCR Clean-up beads. cDNA fragmentation, end-repair, A-tailing and ligation of sequencing adaptors was performed according to manufacturer specifications. The final library was quantified with the Qubit HS DNA kit and the fragment size was analyzed using an Agilent Fragment Analyzer system. Libraries were pooled and sequenced on an Illumina NovaSeq to generate 50,000 reads per cell with a sequencing configuration of 28 base pair (bp) on read1 and 98 bp on read2. Isolation of single cells was performed in three batches on separate days with samples from each treatment group included each day to mitigate batch effects.

Fecal samples were self-collected using a stool sampling kit within 24 hours of scheduled clinic visits and delivered refrigerated or frozen to the clinic coordinator. Once returned to the clinic coordinator, samples were stored at −80 °C until analyzed. Stool samples were subsampled with sterile cotton swabs. Fecal DNA was extracted using FastDNA® KIT (MP Biomedical; Santa Ana, CA; cat#116540400) following manufacturer’s instructions and including additional wash steps. Quantification and dilution of isolated DNA PCR was pooled for library preparation. Sample DNA was stored at −20 °C prior to generation of sequencing libraries.
Sequencing libraries were constructed by PCR amplification of the V4 region of the 16s rRNA gene using primers 515F and 806R following the protocol for the Earth Microbiome Project (http://www.earthmicrobiome.org/protocols-and-standards/16s/). Amplicons were purified using AxyPrep Mag PCR clean-up beads (Axygen; Corning, NY) quantified with Quanti-iT PicoGreen,dsDNA Assay Kit (Invitrogen; Eugene, OR), and pooled in equimolar ratios prior to sequencing at the Colorado State University Genomics Core facility using a 2 × 250 MiSeq flow cell (Illumina, San Diego, CA)^{46 ,47 (link)}.

All the biotinylated primers are placed within 200-bp from the cleavage site. For primer extension, repeated annealing and denaturation for biotin primer (by Sangon, Shanghai) and 20 μg sonicated genomic DNA (0.3–2 kb) was performed as following: 95 °C 3 min; 95 °C 2 min, T_a (annealing temperature) 3 min, 5 cycles; T_a 3 min. Then Bst polymerase 3.0 (NEB) was added to perform primer extension: 65 °C 10 min, 80 °C 5 min. Excessive biotinylated primers were depleted by 1.2× AxyPrep Mag PCR Clean-Up beads (Axygen, US). Purified products were heated to 95 °C for 5 min and then quickly chilled on ice for 5 min for DNA denaturation. Biotinylated PCR products were enriched by Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher).

Biotin primer	Ta (°C)
20–25 nt	50
25–30 nt	55
30–40 nt	58
40–50 nt	60