CXCR4 Gene Sequencing Analysis

After isolation of genomic DNA from CT-2A and GL261 glioma cells using the QIAmp DNA Blood Mini Kit (Qiagen Inc., Valencia, CA), Sanger sequencing of the PCR-amplified products of the CXCR4 gene locus was performed and analyzed by Genewiz LLC (South Plainfield, NJ). Briefly, the two exons of the CXCR4 gene of each glioma cell line were individually amplified by PCR using four pairs of primers: one pair for the first exon and 350-bp upstream region and three pairs for the second exon (Table 1). Each amplicon was purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA) and subsequently sequenced in the forward and reverse directions using the BigDye Terminator Cycle Sequencing Kit (Thermo Fisher Scientific) on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). The sequences obtained were compared with the reference sequences for CXCR4 mRNA and CXCR4 gene locus of C57BL/6J mice (https://www.ncbi.nlm.nih.gov/nuccore; GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"NM_009911.3","term_id":"116268122"}}NM_009911.3 and {"type":"entrez-nucleotide","attrs":{"text":"NC_000067.6","term_id":"372099109"}}NC_000067.6, respectively) using SnapGene software version 2.3.2 (GSL Biotech LLC, Chicago, IL). The threshold for single-nucleotide polymorphism detection was set to 10% of the major peak. All sequencing traces were of quality value >41 (probability of error, <0.010%).

List of PCR Primers and Sequencing Primers Used for Sanger Sequencing of the Mouse CXCR4 Gene

Nucleotides which are underlined indicate noncoding regions.