Effect of CRISPR/Cas9-mediated SYCP1 gene expression depletion in MCF7 breast adenocarcinoma cells (RNA-seq)
Effect of CRISPR/Cas9-mediated SYCP1 gene expression depletion in MCF7 breast adenocarcinoma cells (RNA-seq)
Cell lines were maintained in DMEM media supplemented with 2 mM l-glutamine (Invitrogen) and 10% (v/v) fetal calf serum (FCS) at 37 °C in 5% CO2. Cell lines were never maintained for more than 30 passages or 2 months of continuous culturing. Cell lines were tested for mycoplasma on a tri-monthly basis. SCYP1 knockout was generated through CRISPR/Cas9-mediated genome editing in MCF7 cells and was carried out by Synthego, using a guide RNA (gRNA) targeting exon 2 (5' -AAGCAGCAGTCAGGTGTCTG-3'). Single clones were genotyped using Zero Blunt™ TOPO™ PCR Cloning Kit (Invitrogen, cat. no. 450245) according to the manufacturer's instructions. Upon bacterial transformation, colonies were selected for plasmid isolation using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, cat. no. D4214) and subsequently subjected to Sanger sequencing to identify editing events. At least 5x105 cells were lysed using Trizol reagent, and RNA was extracted with the Directzol™ RNA Mini Prep Kit (Zymo Research, cat. no. R2050) according to the manufacturer's protocol. 1 μg of RNA was used as input for library preparation. rRNA depletion was performed using the NEBNext rRNA Depletion Kit v2 (NEB, cat. no. E7405), followed by cDNA synthesis, end prep, and library amplification using the NEBNext Ultra II Directional RNA Library Prep Kit for 56 Illumina (NEB, cat. no. E7760). The average fragment length was determined using D1000 ScreenTape (Agilent, cat. no. 5067–5582) with D1000 reagents (Agilent, cat. no. 5067–5583), and sample concentration was measured with the Qubit 3.0 Fluorometer (Life Technologies). Next-generation sequencing was performed by Macrogen, Singapore