Shrna2

We have identified two potent shRNAs targeted to CDK2AP1 mRNA. Multiple shRNAs were obtained from commercially available sources (Open Biosystems, PA; Sigma-Aldrich, MO) and screened for their effectiveness. Control scrambled sequences were used similarly. To identify the shRNA clone that produced the strongest knockdown of CDK2AP1, hESCs were transduced with the different shRNA clones using lentiviral vectors and successfully transduced cells were selected by puromycin treatment (1 μg/ml). Following 6 days of selection, antibiotic-resistant colonies were harvested and RNA extracted. QPCR analyses using human CDK2AP1 specific primers were conducted. In our experiments, one shRNA (labeled as shRNA1 henceforth) (Open Biosystems, PA) produced the strongest knockdown and was used in subsequent experiments. For rescue experiments, validated CDK2AP1 shRNA (labeled as shRNA2 henceforth) (Sigma-Aldrich, MO) that target the 3’-UTR was used. In experiments involving analysis of the role of p53, expression was downregulated using lentiviral delivery of p53-specific shRNA (Addgene, MA, USA), followed by validation of knockdown by qPCR analyses.

shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027). Virus-containing medium from HEK293FT cells was collected and used to infect mESCs on two subsequent days. Each day mESCs were incubated in a mix of virus-containing and standard mESC medium (1:1) for 4 hours before changing the medium to standard mESC medium alone. Cells were then washed with cold PBS and collected for analyses.

For lentivirus production, 15 × 10⁶ HEK293T cells were seeded on 15 cm dishes and transfected with VSV-G, pCMVdr8.2dvpr and shRNA plasmids containing specific POT1 knockdown sequences or non-coding shRNA. The transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions. The following plasmids were obtained from Sigma-Aldrich, St. Louis, Missouri: control (SHC016), shRNA1 (TRCN0000039804; predicted knockdown level, 0.56), shRNA2 (TRCN0000010352; predicted knockdown level, 0.92). The plasmids were amplified in maxi cultures and POT1 WT/Q199* DNA was purified using a NucleoBond^® Xtra Maxi EF kit (Qiagen).
HL-60 cells were transduced via spinfection on Retronectin (TakaraBio, Saint-Germain-en-Laye, France) coated 6-well plates. The successfully transduced HL-60 cells were selected with 1 µg/mL puromycin (Invitrogen), which was reduced to 0.5 µg/mL after 1 week. Knockdown of POT1 in HL-60 cells was quantified by qRT-PCR using POT1 TaqMan assays (Hs01565611_m1).