title: GSM8420605: XX_D2_REPi_RE57M_Unsorted_R3; Mus musculus; OTHER
library_construction_protocol: Following cell sorting at the FACS, genomic DNA was isolated using phenol/chloroform extraction. First, cell pellets were incubated for 14 hours at 65 °C in de-crosslinking buffer (1% SDS (Invitrogen), 1.25 µl DTT (Roth) and 10 µl 5M NaCl (Sigma) in Tris-EDTA buffer (Sigma)). Next, 20 µl RNAse A (10 mg/ml, New England Biolabs) were added and the solution incubated for 1 hour at 37 °C. Following, 5 µl Proteinase K (20 mg/ml, Ambion) was added and the solution incubated for 1 hour at 50 °C. Subsequently, 275 µl of Phenol/Chloroform (Roth) were added and the mixture vortexed for 1 min. The samples were then centrifuged for 10 min at 13,000 rpm. The aqueous phase, containing the genomic DNA, was transferred to a new tube and the sample was cleaned using ethanol precipitation. The pellets were dried and resuspended in 50 µl water. The guide cassette was amplified for 20-25 cycles using the KAPA HiFi PCR ReadyMix with primers OG115/OG116. At least 2.2 µg of genomic DNA were amplified per sample. Following clean-up with the DNA Clean & Concentrator Kit, sequencing barcodes were added in a second PCR using the KAPA HiFi PCR ReadyMix for 9-11 cycles