Quick gdna microprep kit

Chicken genomic DNA was extracted from the liver of a Cobb broiler chicken using Quick-gDNA Microprep Kit (Zymo Research, Irvine, CA) according to the manufacturer's recommendations. A series of AvBD9 gene promoter constructs were cloned from chicken genomic DNA using CloneAmp HiFi PCR Premix (Takara Bio USA, Mountain View, CA) with different forward primers paired with a common reverse primer (Table 1). It is noted that the 5′-end of gene-specific reverse primer begins at the third nucleotide upstream of the start codon of the AvBD9 mRNA (GenBank accession number NM_001001611). PCR products were then cloned into a KpnI-linearized luciferase reporter vector, pGL4.21[luc2P/Puro] (Promega, Madison, WI), using a ligation-independent In-Fusion HD PCR Cloning Kit (Takara Bio USA). The presence of the insert in each recombinant plasmid was confirmed with direct Sanger sequencing. Recombinant plasmids were propagated in Stellar E. coli HST08 competent cells (Takara Bio USA) and purified with QIAprep Spin Plasmid Miniprep Kit (Qiagen, Germantown, MD) for transient transfections as described below.

DNA was extracted from plaque samples using the Quick-g DNA MicroPrep kit (Zymo Research, Irvine, CA, USA) and 50 µL (10 mg/ml) of lysozyme per sample to enhance bacterial lysis as described^{54 (link)}. The quantity and quality of DNA was measured spectrophotometrically (Tecan, Männedorf, Switzerland). The primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) which target the hypervariable V4 region of the 16S rRNA gene were used for amplification^{55 (link)}. After, agarose gel electrophoresis was performed to check size integrity. All amplicons were subjected to Illumina MiSeq Platform at the Next-Generation Sequencing Core of University of Pennsylvania and sequenced together at the same run. All Illumina sequence data were submitted to the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA325500.

Genomic DNA was extracted from neocortical tissues using the Quick-gDNA™ MicroPrep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. The quantity of DNA was spectrophotometrically determined at 260 and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). Global DNA methylation changes were measured in neocortical cells at 24 h after treatment using a specific ELISA-based kit (Imprint® Methylated DNA Quantification—Sigma-Aldrich; St. Louis, MO, USA) as previously described [22 (link)]. This kit contained all the reagents required to determine the relative levels of methylated DNA. The methylated DNA was detected using the capture and detection antibodies and quantified colorimetrically using an Infinite M200pro microplate reader (Tecan, Austria). The amount of methylated DNA present in the sample was proportional to the absorbance measured.

DNA from rhesus, cow, horse, dog, squirrel monkey, and mouse placenta tissue was purified using Qiagen's Puregene kit. MethylC-seq libraries were made as described previously [21 (link)]. Briefly, the genomic DNA was sonicated to ~300 bp and methylated Illumina adapters were ligated to the ends. The library was bisulfite converted, amplified for 14 cycles, and sequenced on either an Illumina HiSeq or GAII. For the opossum EEM, rhesus trophoblast, human cord blood, and cow, dog, and opossum brain samples, MethylC-seq libraries were made using the Epicentre EpiGnome Methyl-Seq kit according to the manufacturer's recommendations except that 14 cycles of amplification were performed. For cow oocytes, DNA from 100 cells was purified using Zymo's Quick-gDNA MicroPrep kit and the MethylC-seq libraries were prepared using Zymo's Pico Methyl-seq Library Prep Kit according to the manufacturer's instructions. After sequencing on HiSeq2500/2000 machines, reads were mapped to the respective genomes using BS Seeker [50 (link)] and only one read per genomic position was kept to prevent clonal PCR amplification biases. CpG site methylation data were combined from both DNA strands. Because methylation data was analyzed over large genomic distances and/or smoothed, no minimum coverage was required for CpG sites used in this analysis [35 (link)].

Genomic DNA isolated using the Zymo Research Quick gDNA Microprep kit according to the manufacturer’s instructions was amplified by primers AE7920 and AE7921 using Phusion polymerase to capture the sgRNA-targeted site within PSIP1. Amplified products were assembled with XhoI/EcoRI-digested pIRES2-GFP using NEBuilder HiFi DNA assembly master mix. Recombinant plasmids recovered from at least 10 bacterial colonies per cell clone were sequenced by Sanger sequencing. Predicted amino acid sequences of mutated LEDGF/p75 protein within individual cell clones were deduced from resulting DNA sequences. Such analyses revealed at most two mutant PSIP1 alleles per cell clone.

The DNA from the cell cultures was extracted with the use of Quick-gDNA™ MicroPrep Kit (Zymo Research, Irivine, CA, USA) and quantified spectrophotometrically using the NanoDrop ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The methylation pattern was evaluated using an Imprint Methylated DNA Quantification Kit (Sigma-Aldrich, Saint Louis, MO, USA). According to the manufacturer’s instructions, 50 ng of purified DNA was added to each well where the methylated DNA was detected by capture and detection antibodies. The reaction for color change was monitored using the developing solution. The absorbance of the colorimetric reaction product was measured at 450 nm using an Infinite M200PRO microplate reader.