Quikchange lightning sdm kit

The predicted binding site for miR-96 on the 3’UTR of ABCE1 was PCR-amplified as previously described^{29 (link)} and cloned into the psiCHECK-2 plasmid (Promega). Negative control of ABCE1 was achieved by substituting 3 nucleotides in the seed binding region of the cloned 3’UTRs using the QuikChange Lightning SDM kit (Agilent). HEK-293T and HeLa cells were seeded in 24-well plates supplemented with 10% FBS (GIBCO). Cells were transfected using Lipofectamine 2000 (Rhenium), 5 ng of the psiCHECK-2 relevant clone, 10 ng of pEGFP, and 485 ng miRVec containing the desired pre-miRNA. Twenty-four hours following transfection, lysates were extracted and firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega). The Renilla luciferase results were normalized to the values of the firefly luciferase.

The coding sequences of BCL11A-S (GenBank: NM_138559), BCL11A-L (GenBank: NM_018014), and NONO (GenBank: NM_001145408) were amplified from human fetal brain cDNA using the primers in Table S1 and cloned into pCR2.1-TOPO (Invitrogen). The missense mutations were introduced using the Quik-Change Lightning SDM kit (Agilent) and the primers in Table S2. For expression of fusion proteins with Renilla luciferase, YFP, and mCherry, cDNAs were subcloned into the pLuc, pYFP, and pmCherry expression vectors, respectively, which have been described previously, using the BamHI and XbaI sites.39 (link), 40 For the mammalian one-hybrid assay, a vector for expression of BCL11A fused in frame with the yeast GAL4 DNA-binding domain was created by cutting and re-ligating pBIND (Promega) at the ClaI sites to remove the Renilla luciferase expression cassette. Wild-type and mutant forms of BCL11A-L were subcloned into the BamHI and XbaI sites of this vector. A reporter plasmid was generated by inserting a KpnI-NcoI fragment of pG5luc (Promega) containing five GAL4 binding sites and a minimal adenovirus major late promoter into the vector pGL4.23 (Promega), which contains a codon-optimized firefly luciferase gene. A plasmid containing Renilla luciferase downstream of the herpes simplex virus thymidine kinase promoter (pGL4.74, Promega) was used for normalization.

Construction of the SOD1 minigene, pcDNA_SOD1, and generation of stable minigene cell lines was described previously [20] (link). A splice-defective mutant, pcSOD187M/TO, in which the native U1 consensus sequence, UG GUAAGU was converted to UG GUUGGG to prevent binding of U1 snRNP at the 5′ splice site, was generated by site directed mutagenesis using a QuikChange Lightning SDM Kit (Agilent Technologies) with primers W187F 5′-CATCATTGGCCGCACACTGGTGGTTGGGTTTCATAAAAGGATATGCATAAAAC-3′ and W187R 5′-GTTTTATGCATATCCTTTTATGAAACCCAACCACCAGTGTGCGGCCAATGATG-3 according to the manufacturer’s protocol.
T-REx-293 and T-REx-HeLa cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% fetal calf serum, 0.1 µg/ml streptomycin, 100 units/ml penicillin, and 5 µg/ml blasticidin. Plasmids pcSOD1/TO and pcSOD187M/TO were transfected into T-REx-293 cells using Effectene transfection reagent according to the manufacturer’s protocol (Qiagen). Cells in which the minigene was stably integrated were selected in DMEM media containing 250 µg/ml zeocin. Zeocin-resistant colonies were expanded then tested for induction of expression by tetracycline (TET) using qRT/PCR. Cell lines overexpressing E. coli RNase H were generated as described previously [20] (link).