Plasmids and cell transfection
This protocol is extracted from research article:
RBM5-AS1 promotes radioresistance in medulloblastoma through stabilization of SIRT6 protein
Acta Neuropathol Commun, Jul 5, 2021; DOI: 10.1186/s40478-021-01218-2

RBM5-AS1- and SIRT6-targeting short hairpin RNAs (shRNAs) were synthesized by Beijing Hanyu Biomed (Beijing, China) and cloned to pLKO.1 vector (Sigma-Aldrich). The targeting sequence for RBM5-AS1 and SIRT6 was 5′-GAGUCACAUUCCUUAGCCAUG-3′ and 5′-GACAAACUGGCAGAGCUCCAC-3′, respectively. The RBM5-AS1- and SIRT6-expressing plasmids were constructed by Beijing Hanyu Biomed. All plasmids were verified by DNA sequencing.

Cell transfection was performed using the Lipofectamine LTX Plus (Invitrogen) as per the manufacturer’s protocol. Twenty-four hours after transfection, transfected cells were subjected to gene expression analysis. For selection of stable cell lines, transfected cells were selected in the medium containing 1 μg/mL puromycin (Sigma-Aldrich).

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