Sanger sequencing

Guide RNA targeting the spCas9 endonuclease to regions in the mouse genome encoding Strada (-AGTCGCCATTGGAAGGCCGGAGG-) were calculated in silico using ChopChop software (chopchop.cbu.uib.no). A scramble gRNA (-GACTACCAGAGCTAACTCA-) was used as a transfection and gRNA control. In silico guide RNAs were then assembled into oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA), annealed using ligase buffer (Promega, Madison, WI, USA) at 98°C for 5 min. Annealed gRNA was then sub-cloned into PX330-based plasmid (addgene #48138) using Golden Gate Assembly containing a mCherry reporter linked to Cas9 via a T2a multicistronic element. Plasmid assembly was confirmed by Sanger sequencing (Genewiz, South Plainfield, NJ, USA).
To validate that our gRNA containing CRISPR/Cas9 plasmid created indels in our regions of interest, DNA from Strada, and scramble FAC-sorted cells lines (as described below) as well as wildtype (WT) N2aC was assayed for mismatched DNA pairs (EnGen Mutation Detection Kit; New England Biolabs, Ipswich, MA, USA) with PCR primers targeted towards our genomic region of interest (Integrated DNA Technologies, Coralville, IA, USA).

After the third/fourth round of selection, phage particles were titrated to obtain individual phage colonies, which were used for DNA isolation according to the manufacturer’s protocol for the phage display peptide library.
Single-stranded DNA molecules were used for Sanger sequencing (-96 gIII sequencing primer (5`-CCC TCA TAG TTA GCG TAA CG-3`)) (New England BioLabs, USA). The sequencing reaction products were determined using an ABI 310 Genetic Analyzer (Applied Biosystems, USA) at the Genomics Core Facility of SB RAS. Nucleotide sequences of the inserts encoding peptides were analyzed using MEGA 4.0 software.