title: GSM8420696: XX_D2_TFi_Negative_R2; 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 cycles using the KAPA HiFi PCR ReadyMix with primers OG115/OG116.  To keep 300x coverage, at least 20 µg (TFi screen) or 1.6 µg (TFiMini screen) 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 11-12 cycles