Fri. Jun. 28, 2019 Salmon RRBS library prep

Test out digest and size selection

RRBS DIGEST

Prepared the following reaction 4x with Sample 3 gDNA (418ng/ul):

• 2uL of DNA
• 0.5uL MspI
• 1uL CutSmart

• 6uL H2O

• incubate at 37C 1 hour
• add 0.5uL Taq a1
• incubate at 65C for 30min
• let cool to RT then hold on ice

SIZE SELECTION

PART 1:

• add 6uL beads to each sample (0.6X DNA:Bead ratio), vortex
• incubate at RT 10 min
• magnetic stand for 5 min
• remove and save supernatent
  • • one sample only had 14uL, not 16uL so that means it only had 8uL of digest reaction to start so ratio was actually 0.75X; not 0.6X

PART 2:

• add:
  • 10uL beads (0.75X intial to 2X final)
  • 12uL beads (0.6X initial to 1.8X)
  • 14uL beads (0.6X initial to 2X final)
• vortex samples and incubate at RT 10 min
• magnetic stand 5 min
• remove supernatent
• Wash beads (from part 1 and part 2) with 500uL 80% EtOH
• Repeat wash 1X
• Let beads dry 2-5min at RT (until EtOH smell gone)
• Resuspend beads in 12uL elution buffer (preheated to 50C)
• Incubate at RT for 10 min
• Bind beads 5 min
• Transfer supernatent to clean tube
• measure concentration of part 2 s/n
  • 0.75X to 2X = 3.74ng/ul = 45ng yield
  • 0.6X to 1.8X = 8.8ng/ul = 105ng yield
  • 0.6X to 2X = 10ng/ul = 120ng yield
  • STD2 = 9.98ng/ul

Load gel:

Lanes:

1. Ladder (o’generuler 100bp-1kb ladder)
2. 0.75X to 2X elution from part 1 (large frags excluded)
3. 0.75X to 2X elution from part 2 (size selected frags)
4. 0.75X to 2X supernatent from part 2 (small frags excluded)
5. 0.6X to 1.8X elution from part 2 (size selected frags)
6. 0.6X to 2X eluction from part 2 (size selected frags)
7. 0.6X to 1.8X supernatent from part 2 (small frags excluded)
8. 0.6X to 1.8X elution from part 1 (large frags excluded)

CONCLUSIONS:

• 1ug of DNA yields enough for the library prep after size selection
• Don’t need to digest overnight; maybe digest for 2 hours with MspI and 1 hour for Taq a1; also will be using more enzyme in real RRBS run (30U and 60U, respectively). I only used 10U here
• DNA:Bead ratios for size selection:
  • 0.75X to 2X gives range closest to 100-300bp
  • 0.6X keeps too many fragments > 300
  • 1.8X final ratio removes too many fragments around 100bp
  • If I do the lower selection 2x, I’ll have a better chance of removing small fragments

PLAN:

RRBS DIGEST

DNA: aliquot 1ug of DNA and add nanopure water up to 20uL so all DNA is 1ug in 20uL final vol.

Prepare the following reaction mix:

Add 10uL reaction mix to each tube of 20uL of DNA for 30uL total rxn vol.

37C 2 hour

Prepare the following reaction mix:

add 10uL reaction mix to each tube (now 40uL total rxn vol.)

65C for 1 hour

let cool to RT then hold on ice

SIZE SELECTION

PART 1:

• add 30uL beads to each sample (0.75X DNA:Bead ratio), vortex
• incubate at RT 10 min
• magnetic stand for 5 min
• keep supernatent

PART 2:

• add 50uL beads (0.75X intial to 2X final)
• vortex samples and incubate at RT 10 min
• magnetic stand 5 min
• remove supernatent
• Wash beads (from part 1 and part 2) with 500uL 80% EtOH
• Repeat wash 1X
• Let beads dry 2-5min at RT (until EtOH smell gone)
• Resuspend beads in 10uL elution buffer (preheated to 60C)
• Incubate at RT for 10 min
• Bind beads 5 min
• Transfer supernatent to clean tube

second purification step

• add 20uL beads to each tube
• vortex samples and incubate at RT 10 min
• magnetic stand 5 min
• remove supernatent
• Wash beads (from part 1 and part 2) with 500uL 80% EtOH
• Repeat wash 1X
• Let beads dry 2-5min at RT (until EtOH smell gone)

• Resuspend beads in 21uL elution buffer (preheated to 60C)

• measure concentrations