DAP-seq pipeline exploration
To Order:
Plasmids: Clone or have produced the Transcription Factor Sequences into pIX-HALO-PaqCI or pIX-HALO plasmids.
pIX-Halo-T7-fwd (5′-GTGAATTGTAATACGACTCACTATAGGG)
pIX-Halo-AfterPolyA-rev (5′-CAAGGGGTTATGCTAGTTATTGCTC)
TnT® Quick Coupled Transcription/Translation System
40 reactions at $859, 5 reactions at $236
Magne HaloTag Beads (Promega G7282)
1 ml at $158 or 5 ml at $642
Genomic DNA library preparation(SeqWell Prep until Amp):
Setup
Thaw 5X Rxn Buffer (orange), Tagmentation Enhancer(yellow), Diluent(red), Read 2 Adapter(blue), UDI primer plate, and MAGwise beads at RT
Place Tnx Read 1 Tagging Reagent(white), Ligase(green), and Amplification Master Mix(clear) on ice
Prepare 80% EtOH, 500 ul per sample
Set out 10 mM Tris-HCl and low EDTA TE
Tagmentation
Vortex TnX Read 1 at 60% vortex speed, Vortex yellow and orange tubes at 90% speed.
Reagent | ul/rxn | Rxns | ul needed |
|---|---|---|---|
TnX Read 1(white) | 8 | 1 | 8 |
Tagmentation Enhancer(yellow) | 2 | 1 | 2 |
5X Rxn Buffer(orange) | 5 | 1 | 5 |
Vortex Tag MM at 70% speed
Add 5 ul TE and 5 ul Lambda DNA at 10 ng/ul for a total of 50 ng
Pipette mix 10+ times at 15ul then quick spin
Run on Thermocycler program XTAG:
37C | 7:00 |
95C | 3:00 |
4C | hold |
Return Tagmentation Enhancer(yellow), TnX Read 1 Tagging Reagent(white), Diluent(red) to the freezer. Keep 5X rxn Buffer on ice. Proceed Immediately To Ligation
Ligation
Prepare MM while XTAG is running. Vortex at 70% speed the green, orange, and blue tubes:
Reagent | ul/rxn | Rxns | ul needed |
|---|---|---|---|
Read 2 Adapter(blue) | 4 | 1.1 | 4.4 |
5X Rxn Buffer (orange) | 3 | 1.1 | 3.3 |
Ultrapure Water | 7 | 1.1 | 7.7 |
Ligase (green) | 1 | 1.1 | 1.1 |
Vortex Ligation MM at 70% speed. Place on ice until XTAG program concludes.
Spin down Tagmentation plate and add 15 ul MM per rxn
Pipette mix 10+x at 15ul. Quick spin and add to thermocycler LIGATE program:
25C | 5:00 |
4C | hold |
Once program completes, proceed immediately to 1st bead cleanup
Post Ligation Bead Clean
Spin down rxn plate
Vortex RT MAGwise beads and add 40 ul (1X) to each rxn well
Incubate 5 Min
Move to Magnet and incubate 5 min on it
Remove and discard supernatant
Add 150 ul 80% EtOH, Incubate 30s, Remove 150ul EtOH
Add 150 ul 80% EtOH, Incubate 30s, Remove 150ul EtOH
Remove from magnet, Spin down, Return to magnet for <30s, Remove remaining EtOH
Remove from Magnet, Add 22ul 10 mM Tris-HCl to each well and resuspend
Incubate 5 Min
Return to magnet and incubate 2 Min, Transfer 20 ul to new plate or well
Freeze at -20C or proceed to DNA affinity purification
Transcription Factor Production
PCR HALO modified TF sequences
Use plasmid and pIX-Halo-T7-fwd and pIX-Halo-AfterPolyA-rev to amplify requisite sequences.
DNA Affinity Purification