Dna clean and concentrator 5 kit

PCR was performed as described above using ~50 ng of gDNA and appropriate primers for each target. Amplicon lengths were designed to approach 250 bp for sequencing. PCR purification was performed using the DNA Clean and Concentrator-5 Kit (Zymo Research) according to the manufacturer’s protocol, with an elution volume of 30 µl. All primer sequences are provided in Supplementary Table 3.

Genomic DNA was isolated from LGG1, LGG2 and LGG2-hTert cell lines using DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following manufacturer’s protocol. Quality and concentration of the DNA samples were examined by NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA) and 25 ng of DNA was used to perform PCR amplification of BRAF exon15 with the following primers: 5’-CCT​AAA​CTC​TTC​ATA​ATG​CTT​GCT-3’ (forward) and 5’- GGC​CAA​AAA​TTT​AAT​CAG​TGG​A -3’ (reverse). PCR conditions were set up according to manufacturer’s instructions of Invitrogen™ Platinum™ II Taq Hot-Start DNA Polymerase (Thermo Fisher Scientific). The amplified products were purified using DNA clean and concentrator™-5 kit (Zymo Research, Irvine, CA) and sequenced in forward and reverse directions on ABI 3730 automated sequencer (Applied Biosystems Inc. Carlsbad, CA, United States).

The P. pastoris strain used in these studies was JC308 (James Cregg), a ura3 auxotroph of the GS115 background strain. All yeast growth was performed at 30°C; all bacterial growth was performed at 37°C. The plasmid vectors used in this study were previously described [38] (link). All E. coli work was done using Alpha-Select Gold Efficiency competent cells (Bioline). All enzymes used were from New England Biolabs unless otherwise noted. Primers were purchased from IDT unless otherwise noted. PCR purification and purification of digested plasmids was done using the DNA Clean and Concentrator-5 Kit (Zymo Research). Plasmid DNA was purified using the Wizard Plus SV Miniprep Kit (Promega).

Plasmids used in this study (Supplementary Data 7) were constructed through Gibson assembly. Gibson assembly was performed by amplifying both the gene of interest and the destination plasmid (pCS952^{68 (link)} or pET28) with 40 bp homologous overhangs. PCR amplifications were performed using Q5 DNA polymerase (NEB) and linear DNA fragments were purified using the DNA Clean and Concentrator-5 kit (Zymo Research). Assembled plasmids were propagated in chemically competent E. coli (TOP10; Thermo Fisher Scientific) using heat-shock transformation and selection on Luria-Bertani (LB)-agar plates with carbenicillin (100 μg/mL; for pCS952 derived plasmids) or kanamycin (50 μg/mL; for pET28 derived plasmids). Plasmid DNA was isolated by alkaline lysis from overnight E. coli cultures grown at 37 °C and 250 rpm in selective LB media using Econospin columns (Epoch Life Science) according to the manufacturer’s protocol.

Genomic regions where a sequence variation was found were amplified using 20 ng of genomic DNA (gastric cancers and corresponding non-cancerous tissues) and primers listed in Supplementary Table S6. The PCR products were purified by a DNA Clean and Concentrator-5 Kit (Zymo Research, Irvine, CA), and were sequenced by using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fischer Scientific) and 3730xl DNA Analyzer (Thermo Fischer Scientific). Sequence variations detected only in gastric cancers were considered as a somatic point mutation. Hotspot mutations were defined using information registered in COSMIC. Namely, a pathogenic mutation at the specific base position whose frequency was 5% or more of all the mutations in a specific gene was defined as a hotspot mutation. Among the 154 variations detected in 72 gastric cancers (newly analyzed cases in this study) by a next-generation sequencer, 101 variations were confirmed by Sanger sequencing (54 and 47 were somatic mutations and SNPs, respectively).

About 50,000 cells were resuspended in cold ATAC-seq resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2). Cell nuclei were then prepared by incubation in 50 μl of ATAC-seq resuspension buffer containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin on ice for 3 min. After centrifugation, nuclei were resuspended in 50 μl of transposition mix (25 μl 2× TD buffer, 2.5 μl Nextera Tn5 transposase (Illuminar), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water), and incubated at 37 ^oc for 30 min in a thermomixer with shaking at 1000 rpm. Transposed fragments were then purified with a Zymo DNA Clean and Concentrator-5 Kit. All libraries showed sufficient amplification after the five pre-amplification cycles and were quantified using the KAPA Library Quantification Kit. Libraries were then sequenced using Illumina Novaseq 6000 at the Duke sequencing core. All samples were performed in biological replicates.

One to four liters of samples were filtered through a 0.2 μm Sterivex filter cartridges (EMD Millipore, Billerica, MA). Filters were flash frozen in liquid nitrogen and stored at −80°C until processing. DNA extraction followed previously described protocols (Huber et al., 2002 (link); Sogin et al., 2006 (link)). DNA extracts were purified with the DNA Clean and Concentrator™-5 kit (Zymo Research, Irvine, CA) according to the manufacturer's instructions. DNA extracts were quantified using a Qubit® 2.0 Fluorometer (Life Technologies, Grand Island, NY, USA).