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:

1 rxn 21 rxns reagent
20uL DNA
1.5uL 31.5uL MspI
3uL 63uL CutSmart
5.5uL 115.5uL H2O

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

37C 2 hour

Prepare the following reaction mix:

1 rxn 21 rxns reagent
2.5uL 52.5uL Taq a1
1uL 21uL CutSmart
6.5uL 136.5uL H2O

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
Written on June 28, 2019