RNA-seq of HNSCC cell lines with over-expression and knockdown of MMP10
To investigate MMP10 downstream targets involved in the regulation of tongue cancer metastasis, transcriptome sequencing of MMP10 overexpression (AW13516) and knockdown clones (AW8507 and CAL27) was performed. 1) For stable overexpression of MMP10 in AW13516 cell line, pBABEpuro-MMP10 construct was used. pBABE-puro empty vector was used as control for overexpression. Transfection was carried out using lipofectamine kit (Cat No. L3000015, Invitrogen) in 293FT cells and virus was collected after 48 hours. AW13516 cells were infected with viral particles to generate stable MMP10-overexpression or vector control clones. Stable MMP10-overexpression clones generated and used for RNA-seq are: a) AW13516-vector control; b) AW13516-MMP10 overexpression clones. 2) For shRNA mediated stable knockdown of MMP10 in CAL27 and AW8507 cell lines, 3 lentiviral shRNA constructs targeting MMP10 were used. A short hairpin non-targeting (sh-NT) construct was used as vector control. Transfection and infection with viral particles was performed as described in the overexpression study. Stable MMP10-knockdown clones generated and used for RNA-seq are: a) AW8507 shRNA-NT vector control, b) AW8507 MMP10 shRNA-1, c) AW8507 MMP10 shRNA-2, d) AW8507 MMP10 shRNA-3, e) CAL27 shRNA-NT vector control, f) CAL27 MMP10 shRNA-1, g) CAL27 MMP10 shRNA-2, h) CAL27 MMP10 shRNA-3. To identify the differentially expressed genes, cuffdiff and NOISeq tools were used, and overlapping significantly differentially expressed genes (-1.5<log2FC>1.5; p-value<0.05 or prob>0.95) were identified for further characterization.