GSM9571590: W326R_S4_dedup_assembled_clones; Mus musculus; OTHER
Genomic DNA was isolated as described (PMID: 31230816) with minor modifications. Equal volume 2× lysis buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 4% SDS and 200 µg/mL Proteinase K, New England Biolabs, P8107S) was added to cell suspensions, followed by 30 min incubation at 55°C with agitation (700 rpm). Next, RNase A (Thermo Fisher Scientific, EN0531) was added at 20 µg/mL, followed by 30 min incubation at 30°C with agitation. After phenol-chloroform extraction, ethanol precipitation was performed, using linear polyacrylamide (Thermo Fisher Scientific, J67830.XF) as a carrier. Libraries were prepared as described (PMID: 29725123), modified for analysis of VH recombination only. DNA was sheared to 500bp (Covaris E220 Focused-ultrasonicator), and processed with NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, E7546). For subsequent adapter ligation, 30 µL of Ligation Master Mix and 1 µL of Ligation Enhancer, along with 1.25µL P7 adapter F1 R1 mix (15µM) and 1.25µL P7 adapter F2 R2 mix (15 µM), were added to the previous 65µL reaction, followed by 2 h incubation at 20°C. Adapters were made with P7 adapter F1, P7 short adapter R1-block, P7 adapter F2 and P7 short adapter R2-block. Clean-up was performed with 1X volume Agencourt AMPure XP beads (Beckman Coulter, A63881), eluting the DNA in 25µL EB (10mM Tris-HCl, pH 8.5). Primer extension was then performed in 50µL reactions using Vent (exo-) DNA polymerase kit (New England Biolabs, M0257) and biotinylated J primers (Mouse_JH1_Rev_Bio, Mouse_JH2_Rev_Bio, Mouse_JH3_Rev_Bio and Mouse_JH4_Rev_Bio) as described (PMID: 29725123). Samples were purified using QIAquick PCR Purification Kit and eluted in 40µL EB; primer extension products were captured using 5µL Dynabeads MyOne streptavidin T1 beads (Invitrogen 65601) as described113, and bead-captured products resuspended in 10.5µL EB. PCR amplification then incorporated P5 adapter using P7 short adapter primer and J-P5 short primer mix (25µL reactions, Q5 High-Fidelity 2x Master Mix; New England Biolabs, M0492S; 19 cycles). After PCR, Dynabeads were removed, AMPure XP clean-up performed, and PCR products eluted in 10.5µL EB. Next, incorporation of flow-cell-binding and barcoding sequences was performed by PCR (25µL reactions, Q5 High-Fidelity, 6 cycles) with P7 flow cell and P5-I5 flow cell index primers. P5-I5_flow_cell_index_2, 5, 6, 12, 15 and 19 were used to barcode our six samples. PCR products were purified using two tandem AMPure XP beads clean-up steps, using 15µL for final elution. Barcoded sub-libraries were quantified using Agilent High Sensitivity DNA Assays on an Agilent 2100 Bioanalyzer. Equimolecular amounts of each were pooled to create a final library for paired-end sequencing (2x300bp; Illumina MiSeq).