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=Agilent SureSelect Human Allexon capture= ==Overview and time requirements== [[File:agilent sureselect protocol.jpg]] {| {{table}} | align="center" style="background:#f0f0f0;"|'''Step''' | align="center" style="background:#f0f0f0;"|'''Time''' |- | gDNA library production||1 day |- | library hybridization||24-72 hrs |- | bead preparation||30 minutes |- | capture selection and washing||2 hours |- | DNA purification||30 minutes |- | Post-hybridization amplification||1 hour |- | PCR purification||30 minutes |- | size selection and purification||4 hours |- | |} ==gDNA sample preparation== *Agilent protocol uses Preparing Samples for Paired-End Sequencing (Illumina), our protocol uses NEBNext DNA sample Prep Master Mix Set 1 (NEB, E6040S/L) #End-Repair reaction (blunt end sample with 3' phos.) #Qiagen Qiaquick column purification #Add ‘A’ Bases to the 3' end of the DNA fragments #Qiagen Qiaquick column purification #ligate the single-end adapters #Qiagen Qiaquick column purification (Ampure DNA purification kit can also be used) #amplify adapter-ligated samples #Qiagen Qiaquick column purification and PAGE gel verification *previous preparation steps can be found under labnotes 1-10-2010 for DF and foreskin sets *previous preparation steps can be found under labnotes 1-15-2010 for CViB and CViF sets *DF and foreskin sets still have purified post-PCR product after ligation *ligation step and beyond for CViB and CViF sets can be found under labnotes 2-15-10 ==library hybridization== #Aliquot 500ng of preped gDNA libraries and concentrate at <45C down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O. #mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes): ##For every reaction, add 25ul of SureSelect Hyb #1, 1ul of SureSelect Hyb #2 (red cap), 10ul of SureSelect Hyb #3 (yellow cap), 13ul of SureSelect Hyb #4, total is 49ul ##For every 4 reactions, add 100ul of SureSelect Hyb #1, 4ul of SureSelect Hyb #2 (red cap), 40ul of SureSelect Hyb #3 (yellow cap), 52ul of SureSelect Hyb #4, total is 196ul #prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps): ##add 5ul of SureSelect Oligo Capture library and 1ul ddH2O ##prepare 1:1 dilution of the RNase Block (purple cap) ##add 1ul of diluted RNase Block to each capture library and mix thoroughly by pipetting ##keep tubes on ice #prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul): ##put 3.4ul of prepped library(500ng) into tube B add 2.5ul of SureSelect Block #1, 2.5ul of SureSelect Block #2, and 0.6ul of SureSelect Block #3 ##mix well by pipetting and cap the tubes ##heat up the prepped libraries + blockers at 95C 5min and 65C hold ##maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries) ##incubate both tubes at 65C for at least 5 min before go on #put the SureSelect Oligo Capture Library from step 3.4 into tube C and incubate at 2 min (now the thermocycler should have three tubes per sample incubating at 65C) #while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample #while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample #mix well by pipetting and the hybridization mix should be around 27-29ul #incubate the hybridization mixture at 65C for 24-72 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr) ==Prepare magnetic beads== #prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect" #resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen) #for each hybridization, add 50ul dynalbead to 1.5ml tube #wash the beads: ##add 200ul of SureSelect Binding Buffer ##mix the beads on a vortex for 5 sec ##put the tubes into a magnetic separator, remove and discard the supernatant ##repeat the above steps (4.1-4.3) for a total of 3 washes #resuspend the beads in 200ul of SureSelect Binding buffer ==Select Hybrid capture with SureSelect== #estimate the volume of hybridization mix that remained after 24-72hr incubation #add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times #incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed) #briefly spin in a centrifuge #separate the beads and buffer on a magnetic separator and remove the supernatant #Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec #incubate the samples for 15 min at RT #wash the beads: ##separate the beads and buffer on magnetic separator and remove the supernatant ##resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec ##incubate the samples for 10min at 65C ##invert the tube to mix as the beads may be settled ##repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed #mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads #incubate the samples for 10 min at RT #separate the beads and buffer on Dynal magnetic separator #transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA #Desalt the capture solution with Qiagen Minelute column and elute in 15ul EB ==post-hybridization amplification and purification== *Prepare the Post-Capture PCR Master Mix in a 1.5ml tube. *Aliquot 5μl of bead-bound captured DNA as template into 3 PCR tubes/wells. Mix well by pipetting up and down 5 times. x3 post-capture DNA: 5ul Phusion High-Fidelity PCR Master Mix (2x): 50μl PCR grade water: 41ul Syb_FP5, 100 μM (Final Conc.: 2μM): 2ul Syb_RP7, 100 μM (Final Conc.: 2μM): 2ul SYBR green 50x: 0.4ul Total : 100ul 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C *Qiaquick column purification and elute in 40ul EB
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