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Required reagents:

» SureSelect Human all Exon Kit: Agilent Technologies, #G3362
» Dynabeads M-280 Streptavidin: Invitrogen, #112-05D
» Nuclease-free water, not DEPC treated: Ambion
» MinElute PCR purification kit: Qiagen, 28004

[править] Library Hibridization

  • it is unpractical to process more than 8 samples in parallel
  • it is necessary to have 500ng of amplified library in 3.4µl of EB

[править] Prepare

  • set up heated lid on the thermal cycler at 105°C[1]

  • sign:
» 0.2ml PCR tubes lib_name: two per one library
» 0.5ml LowBind tube TEM: "Target Enrichment Mixture"
» tubes: 0.2ml PCR tubes if total volume is less than 80µl. Otherwise 0.5ml LowBind tube for mixture preparation and 0.2ml PCR tubes for <80µl aliquots:
HB: "Hibridization buffer"
SCL: "SureSelect Capture Library mix"

  • prepare hybridization buffer for all libraries in 0.5ml HB[2]
» vortex, spin down
» (if necessary) prepare <80µl aliquots in 0.2ml PCR tubes
» keep on room temperature

1 hyb.2 hyb.
SureSelect Hyb#112.5µl15.2µl
SureSelect Hyb#20.5µl0.6µl
SureSelect Hyb#35.0µl6.2µl
SureSelect Hyb#46.5µl8.0µl

  • prepare "SureSelect Oligo Capture Library Mix" for all libraries in 0.5ml SCL
» vortex, spin down
» (if necessary) prepare <80µl aliquots in 0.2ml PCR tubes
» keep in ice bath

1 hyb.2 hyb.
SureSelect Oligo Capture Library5.0µl10.7µl
Nuclease-free water1.5µl3.2µl
RNase block0.5µl1.1µl

  • prepare "Target Enrichment Mixture" for all libraries in 0.5ml TEM
» vortex, spin down
» keep in ice bath

1 hyb.2 hyb.
SureSelect Block #12.5µl5.2µl
SureSelect Block #22.5µl5.2µl
SureSelect Block #30.6µl1.3µl

  • put libraries in 0.2ml PCR tubes lib_name: 3.4µl

  1. add to each library 5.6µl of "Target Enrichment Mixture" (TEM);
  2. put library tubes in the thermal cycler;
  3. run the program (be sure, that lid on the thermal cycler at 105°C)
    1. 95°C, 5:00
    2. 65°C, 0 / hold

  4. when the temterature drop down to 65°C
    » put HB and SCL 0.2ml tubes in thermal cycler, keep them at 65°C for a minimum 10 min.
    » put second set of 0.2ml lib_name tubes in thermal cycler
    » set up heated lid on the thermal cycler at 85°C[1]

  5. for each library on termal cycler:
    » prepare 200µl pipette settled on 35µl with a new tip
    » add 13µl of "Hibridization buffer" (HB) and mix
    » add 7µl "Capture Library mix" (SCL)
    » with 200µl pupette mix (slowly pipetting up and down 6x times) and transfer mixture into a new lib_name tube

  6. incubate the hybridization mixture for 24 hours at 65°C

[править] Hybrid capture

  • should be done in advance upon arrival of the Dynabeads M-280 Streptavidin:
» vortex beads (2-5 min)
» aliquot 50µl of beads into 1.5ml LoBind tubes
» store at 4°C

  • prepare
» 65°C water bath
» 50µl aliquots of Dynabeads: one per library
» sign 1.5ml LowBind tubes
lib_name: enriched library
REST: lib_name: the rest of the library

  • check that you have enough buffers:

1 hyb.
Binding buffer800µl
Wash buffer #1500µl
Wash buffer #21.5ml

  • put "Wash Buffer #2" in 65°C water bath until you need it;

  • prepare magnetic beads
  1. vortex beads ~1 min.;
  2. ○○○   three times "Binding buffer" wash:
    • add 200µl of Binding buffer
    • vortex (30 sec), pulse-spin
    • magnetic rack (1 min), remove supernatant
  3. resuspend Dynabeads in 200µl of "Binding buffer";
  4. sign tubes with Dynabeads: lib_name

[править] Hybrid capture

  1. binding with Dynabeads:
    • spin down hybridization mixture and transfer it to signed tube with Dynabeads
    • mix carefully by fingertyping
    • rotate 30 min. at room temperature[3]
    • pulse spin
    • magnetic rack (1 min), transfer supernatant into 1.5ml tube REST: lib_name[4]
  2. washing with "Wash Buffer #1":
    • add 500µl of "Wash Buffer #1"
    • vortex (10 sec)
    • rotate 15 min. at room temperature[3]
    • magnetic rack (1 min), transfer supernatant into 1.5ml tube REST: lib_name
  3. ○○○   three times "Wash Buffer #2" wash:
    • add 500µl of prewarmed "Wash Buffer #2"
    • vortex (5 sec), pulse-spin
    • incubate for 10 min. at 65°C in water bath
    • magnetic rack (1 min), remove supernatant
  4. library elution:
    • resuspend the beads in 50µl of "Elution buffer"
    • rotate 10 min. at room temperature[3]
    • magnetic rack (1 min), transfer supernatant into 1.5ml tube lib_name[5]
    • add 50µl of "Neutralization Buffer" to the collected supernatant
  5. purify library on silica spin column:
    • add 500µl of PB
    • purify MinElute spin column
    • elute with 2x 9µl EB
  6. should be 16µl, snap freeze, store at -20°C

[править] Library amplification and analysis

» Illumina RT PCR: #603 and #611 primers: complexity should be 1-6x107 per 1µl
» large-scale PCR:
SureSelect GA PCR Primers or
"PE_PCR primer 1.1" and "PE_PCR primer 2.1"

[править] Notes

  • Dynabeads adsorbs some DNA w/o enrichment probe. In three controls (library "enrichment": hybridization w/o probes) background level was ~1/2000 if compare with hibridisation with probe.

  1. 1,0 1,1 SETUP ⇒ Lid ⇒ Mode ⇒ constant ⇒ Lid target ⇒ °C
  2. if precipitate forms in hybridization buffer, warm it at 65°C for 5 min.
  3. 3,0 3,1 3,2 10rpm
  4. library w/o captured DNA fragments
  5. captured DNA fragments
Источник — «http://zbio.net/wiki/Enrichment»

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