Arichard:Notebook/2013/December

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December, 2012[edit]

12/02/2013[edit]

Meeting with Ben Siddoway and Gwen Kaeser.

Discussed possible quality control measures.

  • Neuronal nuclei come from three brain banks:
    • UC Irvine
    • Maryland
    • Wash U

12/03/2013[edit]

Shadowed Gwen for neuronal nuclei isolation

[Protocol used]

We decided to use the same samples, but take samples along the way for bulk prep (Nextera). Female preferred so that presence of Y chromosome can be used as a marker for contamination by myself.

Bulk sorted 3 types, all NeuN+

  • 1864+, female Down's from ???.
  • 1568+, female AD from Maryland.
  • 61788+, female AD from Wash U. (New patient)

Notes

  • Started with 0.095 g 1568, 0.14 g 61788, and 0.3 g 1864.
  • Filtered with 40 um BD Falcon filter.
  • Fixed 25 min, not 30.

Time point samples (3) for bulk prep on each sample (3) (9 total):

  • 1 = After filter
  • 2 = Before FACS
  • 3 = After FACS

All samples were stained with PI instead of DAPI due to concerns about the DNA damage upon excitation. This was unnecessary, however, as DAPI uses a 405 nm Ex wavelenth, which is not UV and nowhere near the absorption peak for thymine dimerization (254 nm).

12/11/2013[edit]

  • Lysed cells in Rui's hypotonic lysis buffer

Hypotonic lysis buffer: Make 1M MgCl2 solution, filter with 0.22um filter Make 10ml 10X NP40 lysis buffer [100mM Tris.HCl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004] Make 1ml 1X lysis buffer + 40ul protease (0.2Au/ml final)

Controls introduced at first step. Positive control was 1 ul of 5.6 ng/ul E. coli gDNA. Negative control was NTC.

  • Digested 3 hours at 50 degC.
  • EtOH precipitated and resuspended in 7 ul H2O. Some cells were very large, leading to excess volume. Of these, only 7 ul was used for Nextera.
  • Followed standard protocol from then on:
    • 7 ul sample, 2 ul 5X HMW buffer, 1 ul 1:50 Nextera. 5 min @ 55 degC.
    • Protease
    • Exo-
    • PCR with adapters
    • PCR with primers
    • Ampure XP
    • Size select and gel extract
    • EtOH precip

File:2013 12 11 bulk nxtpcr.jpg

File:2013 12 11 bulk precut1.jpg

File:2013 12 11 bulk precut2.jpg

File:2013 12 11 bulk cut1.jpg

File:2013 12 11 bulk cut2.jpg

Libraries submitted to Alan:

  • 7 pools
    • 1: AR_bulk_25,27_Dec11
    • 2: AR_bulk_26_Dec11
    • 3: AR_bulk_28,30_Dec11
    • 4: AR_bulk_29_Dec11
    • 5: AR_bulk_31,36_Dec11
    • 6: AR_bulk_32_Dec11
    • 7: AR_bulk_37,38_Dec11