2000bp
Methylation filtration to enrich gene sequences of SP70-1143-2000 bp libraries
For each library, 3-5 ug of genomic DNA was sheared using Covaris S220 Adaptive Focused Acoustics ultra sonicator. Libraries were constructed following standard protocol using the NEBNext DNA Sample Prep Master Mix Set 1 (NEB E6040) and Illumina-compatible paired-end adaptors. The library was digested with McrBC endonuclease (NEB M0272) in a 100 ul reaction containing 30 units McrBC, 1xNEBuffer 2, 200 ug/ml BSA and 1 mM GTP. After 8 hr incubation in 37 C, additional 30 units of McrBC and 1 ul of GTP were added and incubation was continued for another 8 hrs. After the incubation, digested (Methyl filtered –MF) library was purified using the QIAquick PCR Purification kit (Qiagen 28104), run on 2% MetaPhor®agarose (Lonza 50108) gel and fragments of desired sizes were excised from gel, purified with the QIAquick PCR Purification kit and amplified with 15 cycles of PCR using Phusion polymerase (part of the NEBNext kit) and cycling parameters described in the NEBNext kit manual. DNA concentrations were quantified on a Bioanalyzer (Agilent), diluted to 10 nM and loaded on flow cells to generate clusters.
2000bp
Universidade Federal do Rio de Janeiro
2000bp