Monarch pcr dna cleanup kit 5 μg

A SNP (rs30424206)-containing region within the mouse Pten gene was amplified and sequenced as described in Weber et al.^{67 (link)}. Briefly, genomic CRISPR/Cas9 target regions were amplified with Q5^® High-Fidelity DNA Polymerase (New England Biolabs) using PCR primers listed in Supplementary Data 9. PCR products were purified with the Monarch^® PCR & DNA Cleanup Kit (5 μg) (New England Biolabs). For library preparation, end repair and A-tailing was performed (NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^®, New England Biolabs) and an Illumina paired end adapter was ligated. Individual samples were barcoded with eight cycles of PCR. Barcoded samples were pooled and quantified with qPCR. The single pool was sequenced on the Illumina MiSeq (300 bp, paired end). Raw reads were preprocessed with Trimmomatic v0.36 with the following parameter settings: leading/trailing Phred quality score cut-off: 25; minimum read length: 50 nt; minimum average Phred quality score within a sliding window of 10 nt: 25. Forward and reverse reads passing these filters were combined using Flash v.1.2.11. Merged reads were mapped to the mm10 mouse reference genome following variant calling with BBMap (https://sourceforge.net/projects/bbmap). Only variant positions with a coverage of at least 100 reads were considered for downstream analyses.

MiRNA sequencing was performed at the Research Center of the CHU de Québec-Université Laval, Quebec, Canada. Twenty-four libraries for TOC samples (pooling of two replicates from each treatment group) to give three replicates per group and twelve libraries for EV samples to have three replicates within each group were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, USA), according to manufacturer’s instructions. The total RNA from each sample was sequentially ligated to 3′ and 5′ small RNA adapters using T4 RNA ligase (New England Biolabs, Ipswich, MA, USA). Next, cDNAs were synthesized through reverse transcription using ProtoScript II Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) and amplified by PCR. Clean up and size selection of fragments was made using the Monarch PCR & DNA Cleanup Kit (5 μg) (New England Biolabs, Ipswich, MA, USA) and 6% polyacrylamide gel. Finally, the RNA libraries were sequenced (50 bp single-end) on an Illumina HiSeq 2500 platform (rapid mode) (Illumina, San Diego, CA, USA).

All reagents and chemicals used were of analytical grade or HPLC grade. CA125 Native antigen from human ascites was purchased from MyBiosource, USA. Tetra chloroauric acid was purchased from Sigma-Aldrich, India. Monarch PCR & DNA clean up kit (5 μg) and Monarch DNA gel extraction kit was purchased from New England Biolabs, India. Hot start Taq Polymerase was procured from Thermo Fisher Scientific, and all membranes were purchased from MDI, India.