title: Capture sequencing of mus musculus: mTSG brain 8
design: Targeted capture sequencing probes were designed as following: the predicted cutting sites (3bp 5 of PAM) of the 280 gene-targeting sgRNAs in the mTSG library plus the Trp53-targeting sgRNA in the vector were retrieved from mouse genome (mm10). The 140bp sequences of the flanking regions of the cutting sites (5- 70bp and 3- 70bp) were retrieved using Bedtools. The regions were consolidated using NimbleDesign (Roche / NimbleGen), and probe matches were set with these parameters (Preferred Close Matches = 3, where initial selection of probes for a given region will only include probes with 3 close matches or less; and Maximum Close Matches = 20, where if there are insufficient probes available for a given region at the Preferred Close Match number, the threshold will be incrementally increased to 20 until adequate coverage is achieved. After consolidation, a number of 178 regions covering 277 sgRNAs, with a total of 33638bp were covered in the probeset, with Target Bases Covered = 32239 (95.8%) and one target sgRNA without coverage due to a lack of qualified candidate probes in the region. The mTSG-Amplicon targeted capture sequencing probes were synthesized using the SeqCap EZ Probe Pool synthesis procedure (Roche). The capture sequencing was done following standard Illumina-Roche-Illumina protocols. Genomic DNA samples from mouse organs were subjected to fragmentation, followed by a library preparation step using KAPA Library Preparation Kit (Illumina). The libraries were then amplified using LM-PCR, hybridized to the mTSG-Amplicon probe pool, washed and recovered, and amplified with multiplexing barcodes using LM-PCR. The multiplexed library was then QCed using qPCR, and subjected to high-throughput sequencing using the Hiseq-2500 platforms (Illumina) at Yale Center for Genome Analysis. 277/278 (99.6%) of unique targeted sgRNAs were captured for all samples from this experiment, with the missing one being Arid1a-sg5.
library_name: mTSG brain 8