Sanger sequencing of ABLIM1 transcript variants in hCMEC/D3 cells
This study identifies ABLIM1 transcript variants 15 and 16 expressed in the hCMEC/D3 human cerebral microvascular endothelial cell line by Sanger sequencing. Initial screening using six F1-F6/R1 primer pairs showed amplification only with the F1R1 primer pair, which covers 15 known transcript variants. Three subsequent PCR rounds were performed. A first-round PCR using the F/R primer pair yielded three bands of approximately 600, 800, and 900 bp, designated as S1-1, S1-2, and S1-3, respectively. The 800 bp product (S1-2) was sequenced and confirmed to match transcript variants 15 and 16. Based on expected product sizes, the 600 bp band (S1-1) was predicted to correspond to variant 23, and the 900 bp band (S1-3) to variants 4/11/12/27. To determine whether any of the predicted candidates (variants 23, 15, 16, 4, 11, 12, and 27) were actually expressed, a second-round PCR using six primer pairs targeting these seven candidate variants was performed; this confirmed specific amplification only for variants 15 and 16, excluding the presence of the other candidates. A third-round PCR using primers 15-16F/15-16R spanning the 21-nucleotide variable region produced two distinct products, which were gel-purified and designated as S3-1 and S3-2; bidirectional Sanger sequencing confirmed these as variants 15 and 16, respectively. These five gel-purified products (S1-1, S1-2, S1-3, S3-1, S3-2) were subjected to bidirectional Sanger sequencing, resulting in 10 raw .ab1 files.
This study identifies ABLIM1 transcript variants 15 and 16 expressed in the hCMEC/D3 human cerebral microvascular endothelial cell line by Sanger sequencing. Initial screening using six F1-F6/R1 primer pairs showed amplification only with the F1R1 primer pair, which covers 15 known transcript variants. Three subsequent PCR rounds were performed. A first-round PCR using the F/R primer pair yielded three bands of approximately 600, 800, and 900 bp, designated as S1-1, S1-2, and S1-3, respectively. The 800 bp product (S1-2) was sequenced and confirmed to match transcript variants 15 and 16. Based on expected product sizes, the 600 bp band (S1-1) was predicted to correspond to variant 23, and the 900 bp band (S1-3) to variants 4/11/12/27. To determine whether any of the predicted candidates (variants 23, 15, 16, 4, 11, 12, and 27) were actually expressed, a second-round PCR using six primer pairs targeting these seven candidate variants was performed; this confirmed specific amplification only for variants 15 and 16, excluding the presence of the other candidates. A third-round PCR using primers 15-16F/15-16R spanning the 21-nucleotide variable region produced two distinct products, which were gel-purified and designated as S3-1 and S3-2; bidirectional Sanger sequencing confirmed these as variants 15 and 16, respectively. These five gel-purified products (S1-1, S1-2, S1-3, S3-1, S3-2) were subjected to bidirectional Sanger sequencing, resulting in 10 raw .ab1 files.