Accuprime pfx supermix

DNA was prepared for 16S rRNA gene V4 region sequencing exactly as described previously (Kozich et al., 2013 (link)). Briefly, the V4 region of the 16S rRNA gene was amplified individually from each sample with AccuPrime Pfx SuperMix (Invitrogen, Carlsbad, CA) using a dual-indexing strategy as described previously (Kozich et al., 2013 (link)) (Supplementary Table S2). Samples were normalized using the SequalPrep Normalization kit (Applied Biosystems, Waltham, MA, United States). Sequencing was performed at the BYU DNA Sequencing center, and samples plus 10% PhiX control DNA were sequenced using 2x250bp v2 Illumina sequencing kits on a MiSeq (forage diet and supplement experiments, and the comparison of kit-effects), or at the BYU DNA Sequencing center on a HiSeq 2500 (body condition experiment), all following manufacturer’s recommendations. Sequences were deposited to the National Center for Biotechnological Information’s Short Read Archive under study number SRP116192.

ChIP-seq binding sites for STAT3 were analyzed on the UCSC genome browser (GRCh37/hg19) using the ‘ENCODE Regulation Super-track’. ChIP was performed using the SimpleChIP Kit (CST, 9003). HepG2 cells from four 10 cm dishes were used in ChIP. Cells were starved overnight and then treated with IL-6 (25 ng/ml) for 30 min before harvest. In total, 500 μl of cross-linked chromatin were obtained, and each 100 μl (diluted into 500 μl by adding 400 μl 1X ChIP buffer) was immunoprecipitated with 2 μg of the following antibodies: Normal Rabbit IgG (CST, 2729), Phospho-STAT3 (Tyr705, sc-8059), STAT3 (sc-482) and RELA (sc-372). After determination of the appropriate amplification cycle numbers by qPCR, the IPed chromatins were analyzed by standard PCR with AccuPrime PFX SuperMix (Invitrogen, 12344–040). For ChIP assays on the promoters in reporter vectors, HepG2 cells (6 × 10⁶) were transfected with 10 μg of plasmids (P1, P4, P1-M and P4-M) in 10 cm dishes for two days, and then treated as above. In PCR, a GLprimer2 that binds to the coding sequence of firefly luciferase was used as the reverse primer.

Each pegRNA was designed to harbor BsaI restriction sites at the 5′ and 3′ end. The single-stranded pegRNAs were amplified to produce double-stranded pegRNAs using Accuprime PFX Supermix, according to the manufacturer’s recommendation (Invitrogen, Waltham, MA, USA). The amplified pegRNAs were digested with BsaI-HF restriction enzyme (New England Biolabs, Ipswich, MA, USA) and ligated into Antarctic phosphatase-treated/BsaI-digested pU6-pegRNA-GG-acceptor plasmid. For constructing the PE3 plasmids, the complementary top and bottom sgRNA strands were purchased separately. Each strand was designed to harbor the BbsI restriction site at the 5′ ends. The 5′ end of each strand was phosphorylated by using the T4 polynucleotide kinase (New England Biolabs) and annealed together. The annealed PE3 was ligated into Antarctic phosphatase-treated/BbsI-digested pKLV-U6-gRNA(BbsI)-PGKHygro2AeGFP. Briefly, the ligation was performed using the T4 ligase (New England Biolabs) at 16 °C overnight, followed by transformation into the chemically competent DH5α E.coli strain (New England Biolabs). The presence of ligated pegRNA and PE3 into their respective expression vectors were verified via Sanger sequencing using U6 promoter Fwd primer.

The CD19 cDNA was amplified from 18.81 pre-B cells with CD19-forward and CD19-reverse primers using AccuPrime pfx SuperMix (Invitrogen) and cloned downstream of the CMV promoter in pcDNA3.1-zeo to yield pcDNA-CD19-zeo.
CD19 forward: 5′-ACTGCTAGCGCCACCATGCCATCTCCTCTCCCTGTCT-3′
CD19 reverse: 5′-GGGTCTAGATCACGTGGTTCCCCAAGTC-3′.

A gene encoding the RC maquette was purchased from DNA2.0 in a pJexpress414 vector, and the DH maquette gene was purchased from GenScript in a pET-3a vector. Both genes included an N-terminal His₆ tag followed by a TEV protease cleavage sequence. Invitrogen supplied mutagenesis primers, and mutant plasmids were PCR amplified with AccuPrime^™ Pfx SuperMix (Invitrogen). Amino acid sequences are given in Supplementary Table S1.
Plasmids were transformed into BL21-(DE3) competent cells (New England Biolabs), and proteins were expressed and purified as previously described (Ennist et al., 2022 (link)). Where noted, an additional purification step of high performance liquid chromatography (HPLC) was executed on a Waters reverse-phase HPLC system using a C4 or C18 HPLC column (Grace Davison Discovery Sciences). HPLC-pure samples were lyophilized and resuspended in >6.5 M GdnHCl, refolded by dilution, and purified by size exclusion chromatography (SEC) with Superdex 75 prep grade gel filtration medium (GE Healthcare Life Sciences).