Kapa hifi ready mix

Total RNA was isolated from HEK293T cells using BIO TRI RNA reagent (Bio-Lab Chemicals) and mRNA was captured using Oligo d(T)25 magnetic Beads (NEB) according to manufacturer's protocol. Poly(A)-purified RNAs were taken for library preparation using a derivation of MARS-seq (massively parallel RNA sequencing) as described (17 (link)). Briefly, 10 ng of poly(A)+ RNA was taken for the first reverse transcription reaction using Illumina barcoded RT1 primer. Resultant barcoded cDNA samples were subsequently pooled according to Ct values of a house-keeping gene (GAPDH) (Quality control 1). Pooled cDNA was treated with Exonuclease I (NEB) to remove excess primers followed by second strand synthesis. After that, in-vitro transcription was performed using T7 RNA Polymerase (NEB) to generate RNA, which was later fragmented and ligated to an adaptor consisting of RD2 using T4 RNA ligase I (NEB) followed by the second reverse transcription reaction. The library so formed was amplified using Kapa Hifi ready mix (Roche). The amplified RNA libraries were sequenced using a high-throughput 75 bp kit (Illumina FC404-2005) on NEXTseq 500 sequencer.

CAN1 was amplified from gDNA in 6 overlapping, 349-350 base pair fragments using primers listed in Supplementary Table S2. KAPA HiFi ReadyMix (Roche) was used to amplify these fragments in 25 μl reactions for each CAN1 region for each of the 150 samples (total of 906 reactions). Two-microliter of DNA was added to each reaction, in the range of 50–500 ng. Two-microliter of each PCR reaction was electrophoresed on a 1% agarose gel to confirm amplification. For each sample, 20 μl of PCR product from each of the 6 regions (each ∼ 300 bp) were pooled in a 96-well plate and purified using the Zymo ZR-96 DNA Clean-up Kit. A total of 65 pooled sample sets (Supplementary Table S4) were generated for paired-end sequencing (2 × 300), including technical replicates. PCR products from the same genomic preparation of pooled samples were independently amplified and sequenced.

scATAC-seq libraries were prepared using the Chromium Next GEM single-cell ATAC reagent kit v1.1 following the manufacturer’s instructions with the modifications described in the ASAP-seq and mtscATAC-seq protocols to retrieve TotalSeq antibody-derived tags (ADTs), hashtag oligonucleotides (HTOs) and mtDNA^{16 (link)–18 (link)}. The saved eluate from the silane bead elution and the supernatant of the first SPRIselect purification were combined for amplification of ADT/HTO libraries using the KAPA HiFi ready mix (Roche) with sample-specific index primers (Illumina small RNA RPIx/Truseq D7xx) and purified using SPRIselect reagent. Library size and quality were analyzed using a Fragment Analyzer (Advanced Analytical) before fragmentation and after final purification. Final library concentrations were measured on a Qubit 2.0 fluorometer (Thermo Fisher). Libraries were sequenced on a NextSeq500 or NovaSeq6000 sequencer (Illumina) using longer read1/2 configurations than suggested by 10x Genomics to improve mitochondrial genotyping (NextSeq500: R1 72 cycles, R2 72 cycles, I1 8 cycles, I2 16 cycles; NovaSeq6000: R1 88 cycles, R2 88 cycles, I1 8 cycles, I2 16 cycles).

Microbial DNA was extracted from each sample using a ZymoBIOMICS DNA Miniprep Kit (Zymo Research). Shotgun metagenomic sequencing was performed at Washington University GTAC@MGI. The extracted DNA (100 to 250 ng) was fragmented on the Covaris LE220, targeting 375 bp inserts. Automated dual-indexed libraries were constructed using the KAPA Hyper Library Prep Kit (Roche) on the SciClone NGS instrument (Perkin Elmer). The ligated fragments were amplified with KAPA HiFi ReadyMix (Roche) and 8 cycles of polymerase chain reaction (PCR). The amplified libraries were size-selected with a dual-spiral AMPure bead purification, targeting an average final library size of 450 bp. The libraries were assessed on the Caliper GX (Perkin Elmer) to determine their size and mass. Libraries were diluted to 5 nM, and the concentration of each library was validated by performing qPCR using the KAPA Library Quantification Kit (Roche) and the Lightcycler 480 (Roche). Libraries were normalized to 2.5 nM and analyzed using the NovaSeq 6000 S4 300 cycle kit and the XP workflow (Illumina). Approximately 12 GB of 2× 150 paired-end sequence data were generated per sample.

Mutagenesis was performed based on the Quick-change II method as previously described [42 (link)] with the following modifications. Primers were purchased from Integrated DNA Technologies Ltd (Coralville, IA, USA). Kapa HiFi readymix (Kapa Biosystems, Wilmington, MA, USA) or Primestar HS enzymes (TaKaRa Bio, Kyoto, Japan) were substituted for Pfu polymerase. RAMP1 constructs Y66A, H97A and F101A were produced as previously described [43 (link)].

To test the quantity of extracted DNA a spectrophotometer (NanaDrop 1000 Spectrophotometer, NanoDrop Technologies Inc., Thermo Fisher Scientific, Waltham, MA, USA) was used on all extracted DNA samples. The DNA extractions were thawed in rounds of 27 samples and diluted to a range of 30–100 ng/ml. The V4 region of the 16S rRNA gene was amplified using polymerase chain reaction (PCR) with the forward primer 515F(5′GTGCCAGCMGCCGCGGTAA-3′) and reverse primer 806R(5′GGACTCTACHVGGGTWTCTAAT-3′) (Walters et al., 2016 (link)), KAPA HiFi ReadyMix (Kapa Biosystems, Wilmington, MA, USA), and PCR grade water. The PCR products were purified with Mag Bind RXNPure plus (Omega Biotek Inc.). To prepare the purified PCR products for Illumina MiSEq sequencing, the purified PCR products were amplified using Illumina adapters N716-N729 and S513-S522, and then purified again. All finalized PCR products were evaluated using gel electrophoresis and DNA was measured using spectrophotometry to ensure the concentration of DNA was greater than 15 ng/μl before Illumina sequencing.