title: GSM8517836: 3_mousebrain, CT2Amcherry+, AAV8-Control, day 15, rep7; Mus musculus; OTHER
library_name: GSM8517836
library_construction_protocol: 1-2-4) genomic DNA was extracted using DNAeasy DNA isolation kit (Qiagen) and quantified using a NanoDrop (Wilmington, DE) ND-1000. 3) genomic DNA was extracted using the adapted TRIzol™ Reagent Experimental protocol for DNA isolation (Invitrogen, 15596026) from FACS sorted mCherryPOS (tumor cells) and mCherryNEG (non-tumor cells) by utilizing the remaining sample post-removal of the aqueous layer of the in Qiazol lysed RNA (Qiagen). 1-2-4) mmu-miR-21a or off-targets were amplified, using the Phusion High Fidelity PCR kit (New England Biolabs), for Next Generation Sequencing (NGS) analysis primers amplified a 222 bp at a Tm of 67°C DNA fragment of the mouse miR-21. The PCR products were purified using the Qiagen PCR purification kit. Next, the PCR products were loaded on a 2% agarose gel (Bio-Research, Worcester, MA), and DNA fragments were resolved by gel electrophoresis. The purified PCR products were sequenced at the MGH CCIB DNA Core and CRISPR efficiency was determined based on CRISPResso2 analysis 3) Genome modified reads were determined by NGS using a two-step PCR-based Illumina library construction method. The first PCR reaction was done with approximately 100 ng of gDNA using Q5 High-fidelity DNA Polymerase (NEB) and the respective primers, with cycling conditions of one cycle at 98 °C for 2 min; 35 cycles of 98 °C for 10 s, 65 °C for 10 s, 72 °C for 20 s; and one cycle of 72°C for 1 min. PCR-1 products were purified using paramagnetic beads at a ratio of 1.8×. Approximately 20 ng of purified PCR-1 product was used as a template for a second PCR (PCR-2) to add Illumina barcodes with adaptor sequences using Q5 (cycling conditions of one cycle at 98°C for 2 min; 10 cycles at 98°C for 10 s, 65°C for 30 s, 72°C 30 s; and one cycle at 72°C for 5 min). PCR-2 products were pooled based on concentrations from capillary electrophoresis (QIAxcel, QIAGEN). Final libraries were quantified by Qubit dsDNA High Sensitivity assay (Thermo Fisher) and sequenced on a MiSeq sequencer using a 300-cycle v2 kit (Illumina). On-target genome editing activities were determined from amplicon sequencing data using CRISPResso2.
identifiers.namespace: GEO
identifiers.id: GSM8517836_r1