Qiaquick pcr product purification kit

ChIP assay was conducted by using a ChIP assay Kit (Upstate Biotechnology, Billerica, MA, USA) as per the manufacturer’s instructions. Briefly, the cells were harvested, fixed with 1% formaldehyde for 10 min at RT, and resuspended in lysis buffer containing a protease inhibitor cocktail. Following sonication, the soluble chromatin was diluted with ChIP-dilution buffer and immunoprecipitated with C/EBPα antibody or IgG as a negative control overnight at 4° C. Then the chromatin-antibody complexes were eluted with the elution buffer. After reversion of the cross-link, the DNA was purified by Qiaquick PCR product Purification Kit (QIAGEN Inc, Chatsworth, CA, USA). End-point PCR was conducted to amplify the site 1 and 2 (two potential binding motifs for C/EBPα) by using primers spanning the C/EBPα binding elements in the promoter region of the Munc18b gene.

Snails collected from each location were dissected for anatomical identification under a microscope [22 ]. In brief, shells of snails were carefully removed with forceps and the number of prostate diverticula were counted under the microscope as previously described [22 ]. Genomic DNA was further extracted from ~10–30 mg foot tissue of snails examined above using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA sequencing of individual cytochrome c oxidase subunit 1 (cox1) [23 (link)], internal transcribed spacer 1 (ITS1), 5.8S rDNA, and internal transcribed spacer 2 (ITS2), and 16S ribosomal DNA (rDNA) was carried out as previously described [24 (link), 25 ]. All 3 markers were amplified under the same PCR temperature profile: an initial 5 min denaturation step at 94 °C followed by 30 cycles (iterations of 50 s at 94 °C, 50 s at 55 °C and 50 s at 72 °C) plus a final 10 min extension at 72 °C. Gel electrophoreses were performed to confirm successful amplification of the desired fragment of the target size. PCR products were purified using the QIAquick PCR Product Purification Kit (Qiagen, Hilden, Germany). Sequences of purified PCR products were obtained from both directions using the same primer pair for PCR by cycle sequencing in a commercial laboratory (BGI Tech Solutions (Hong Kong) Co., Ltd., Beijing, China).

PCR amplicons were purified by using a QIAquick PCR product purification kit (Qiagen) and quantified by using Qubit (Life Technologies). Eight samples tagged with unique indexes were pooled together at equal quantities to create a single sample for library preparation. Library preparation was performed by using TruSeq DNA library preparation kit V2 (Illumina) without fragmentation. Sequencing was performed on a Miseq bench-top sequencer (Illumina) with 2 × 150 bp methodology. Paired-end sequencing data from the Miseq reporter software was further analyzed off instrument. Quality filtering of the paired end data, de-multiplexing and trimming was performed by using a FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) prior to mapping against reference sequences using Bowtie 2 [11 (link)]. The reference amplicon sequences for data alignment were generated from accession numbers cited by Kong et al. [8 (link)] for serotype-specific capsular gene sequences and [Genebank: AJ419979 and Genebank: AJ244307] for the atypical and typical pneumococcal lytA gene sequences, respectively.