Neuro 2a n2a

HEK293T (American Type Culture Collection; CRL-3216) and Neuro-2a (N2a, American Type Culture Collection; HTB-96) cells were cultured in 10 cm dishes with Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum (v/v) (Gemini) and 1% penicillin–streptomycin at 37 °C with 5% CO₂. Cells were routinely passaged at a ratio of 1:3 at 90% confluency by digesting with 0.25% pancreatin (containing EDTA). During transfection, cells were cultured in 24-well plates in three biological replicates and transfected with 1.3 μg plasmids (including 900 ng PEmax, 300 ng xr-pegRNA, and 100 ng corresponding nick sgRNA) per well, when cells reached an approximate 70% to 90% confluency. Transfections were carried out with the aid of EZ Trans (Life-iLab; catalog no.: AC04L091) reagent and according to the manufacturer′s protocols. For experiments involving WGS or RNA-Seq, HEK293T cells were cultured in larger scale (10 cm dishes). Subsequently, 13 μg of total plasmids for PE-STOP (9 μg PEmax, 3 μg xr-pegRNA, and 1 μg corresponding nick sgRNA), 12 μg total plasmids for iSTOP or i-Silence (9 μg AncBE4max or ABE8e with 3 μg corresponding sgRNA), and 5 μg total plasmids for CTRL group (5 μg pCMV-GFP) per dishes were used for transfections. Cells were transfected at approximately 60% to 90% confluency.

A mouse neuroblastoma cell line, Neuro2A (N2A) (American Type Culture Collection) was maintained in a humidified incubator with 5% CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G and 100 mg/ml streptomycin antibiotics (1%). Individual siRNAs were transfected into cultured cells using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Thermo Fisher Scientific) according to the manufacturer’s protocol in antibiotic-free media. The authenticity of the cell line was validated using short tandem repeat profiling. In addition, the cell line was tested for mycoplasma, and cells were free from mycoplasma contamination for all experiments. Three 60 mm plates were seeded for each experimental condition. Each 60 mm plate was further seeded into three wells in 6-well plates. The 6-well plates were further processed for immunoblotting, DRIP-seq, or RNA-seq. Therefore, each biological replicate for all conditions was subjected to the same analysis. The siRNAs employed were: scramble negative control (Integrated DNA Technologies; 51-01-14-04), EXOSC3 siRNA 1 (IDT; mm.Ri.Exosc3.13.1), and EXOSC3 siRNA 2 (IDT; mm.Ri.Exosc3.13.5). If required, compounds were added to the media: CPT (5 μM, 1 h; Combi-Blocks ST-5997) and bleomycin (25 μg/ml, 3 days; Cayman Chemical 13877).

The wild type human SK-N-SH neuroblastoma cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and used for the generation of our screening cell lines.
The wild type mouse Neuro 2A (N2a) was purchased from American Type Culture Collection (ATCC). All cell lines were cultivated in DMEM Glutamax (Gibco) supplemented with 1% penicillin/streptomycin (Gibco) and 10% inactivated FBS (Sigma-Aldrich), respectively. For detachment, cells were treated with 0.05% Trypsin–EDTA 1x (Gibco) for 10 min at 37 °C. All compounds were purchased from Selleckchem.