Qiaquick gel extraction columns

Tumors were resuspended in Lysis Buffer (10 mM Tris pH 8.0, 10
mM EDTA, 0.5% SDS, 0.75 mg/mL Proteinase K) with a homogenizer, then
incubated at 55°C overnight. Cell pellets were resuspended in
Lysis Buffer without homogenization, and were also incubated at
55°C overnight. gDNA extraction was performed as described above,
for Genome Scale Screens. PCRs were performed using a total mass of 18
ug (across three 100 uL reactions) for cell pellets and 24 ug (across
four 100 uL reactions) for tumors (to compensate for inclusion of
stromal tissue). PCRs were performed with conditions and primers
described above, for Genome Scale Screens. PCR3 products were
gel-purified using QiaQuick Gel Extraction columns (QIAGEN). Samples
were sequenced on an Illumina HiSeq 2500 with the primer heyME19.

Sorted libraries were grown overnight in tryptophan deficient glucose media and 0.5mL culture was used for plasmid DNA extraction using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research D2004). DNA was further purified and concentrated by isopropanol/ethanol precipitation to remove inhibitors. Purified DNA was used to transform MegaX DH10B T1R Electrocomp Cells (Invitrogen C640003) by electroporation. Transformed cells were passaged into 50 mL Luria-Bertani broth with 100µg/mL ampicillin for selection and serial dilution onto Luria-Bertani agar plates with 100µg/mL ampicillin was performed to determine library size. Typical transformations provided a total of 4,000–30,000 colony forming units per library. E. coli cultures were passaged once and 100mL were grown in Luria-Bertani broth with 100µg/mL ampicillin for DNA extraction using two QIAGEN Plasmid Plus Midi columns (QIAGEN #12943). Bacterially derived plasmid DNA was digested with NheI-HF and SalI-HF restriction enzymes (New England Biolabs R3131S & R3138S) to liberate the 633 base pair fragment containing the PrP genes with ablating mutations. DNA fragments were purified on a 2% agarose gel, extracted using 2–4 QIAquick Gel Extraction columns (QIAGEN 28704) and further purified by isopropanol/ethanol precipitation. Final DNA preparations contained 0.5–50 µg total DNA.

Genomic DNA was isolated from HMEC Sublibrary screen pellets
using the GeneJET Genomic DNA Purification Kit (ThermoFisher Cat. #
K0722), according to manufacturer’s instructions. PCRs were
performed using a total mass of 2.6 ug (across four 100 uL reactions).
PCRs were performed with conditions and primers described above, for
Genome Scale Screens. PCR3 products were gel-purified using QiaQuick Gel
Extraction columns (QIAGEN). Samples were sequenced on an Illumina HiSeq
2500 with the primer heyME19.