Gs junior titanium empcr kit lib a

The quantified and purified library pool was then diluted with modified TE-4 (10 mM Tris, 0.1 mM EDTA, pH 8.0) to the concentration of 4 × 10⁵ molecules/uL in order to add a total of 10 uL to achieve a 0.4:1 DNA to bead ratio in emPCR. The optimal DNA to bead ratio was determined to be 0.4:1 based on the number of high quality pass filter reads. Targeting the optimal DNA:bead ratio is critical to avoid the incorporation of more than one molecule of DNA per bead or insufficient bead recovery. The finalized library pool was amplified using the GS Junior Titanium emPCR Kit (Lib-A) (Roche Life Sciences) following the manufacturer’s protocol. All successfully prepared libraries were sequenced on the 454 GS Junior System (454 Life Sciences, Branfod, CT, USA). Amplicon Variant Analyzer (AVA) was used to analyze the 454 sequence data and identify SNP variants compared to the revised Cambridge sequence (rCRS) (20 (link)).

Mice were immunised orally with sheep red blood cells for 2 weeks and intraperitoneally with 10 μg of LPS for 3 days. Single-cell suspensions from Peyer's patches were labelled with B220-APC- (Biolegend, ref:103212), GL7-FITC- (Beckton Dikinson, ref: 561530) and Fas-PE- (Beckton Dikinson, ref: 554258) conjugated antibodies. Purification of B220⁺GL7⁺Fas⁺ cells was realized on a FACS ARIA III (BD). Genomic DNA was extracted, and a region corresponding to a sequence of 517 bp downstream of the J_H4 segment was amplified by PCR. As a control, Igκ light-chain VJ-rearranged fragments were also amplified. Primers (detailed in the Supplementary Table 1) were coupled to 454 Sequencing adaptor sequences and PCR was performed using the program previously reported8 (link). According to the manufacturer, the resulting purified amplicons were prepared for sequencing with a GS Junior Titanium emPCR Kit (Lib-A; Roche), and the library of DNA fragments was sequenced on a 454 GS Junior instrument (Roche). Obtained sequences were aligned to the reference sequence using BWA aligner38 , and SAMtools software was used to obtain BAM files39 (link). Redundant sequences were excluded, and wig files were generated using IGV Tools40 (link) and manually analysed to determine mutation frequencies for each nucleotide in the sequence.

The DNA concentration of the purified amplicons of the on-target and off-target PCR analysis was determined using the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen). To allow discrimination between amplicons from control and CRISPR-expressing cells, we designed primer-sets for the different target areas by adding a 454 linker and a unique barcode to the 5′ end of the primers. All PCR products were diluted to a concentration of 10⁹ molecules/μl and amplicons from gRNA-expressing cells and control cells were mixed in a 2:1 ratio mixed to form the sample library and sequenced using the GS Junior Titanium emPCR Kit (Lib-A) (Roche) and the GS Junior sequencer (Roche). Sequences were analyzed using GS Amplicon Variant Analyzer software (Roche) and MEGA 660 (link). The CRISPR/Cas9 target region was defined as three nucleotides upstream and downstream of cleavage site.