Arichard:Notebook/bulk150210

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February 10, 2015 bulk nextera[edit]

Motivation: I want to make bulk libraries from 1-02 (AD) and 25-01 (CTL) using SM59 with our new IDT-Nextera v2 duel index primers, but I first need to test SM59 against 1:50 Nextera v1 with single index primers. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 to Nextera v1, and wait on the more precious tubes (cortex).

Brandon already tested dilutions of SM59 and EZ-Tn5 (N = 1 per concentration) on HLS lysed cells (for accessibility), but it seems that the variability in lysis might be masking the dilution effect (no clear trend). He did include 6 ng purified genomic, which gave a very bright smear. I will use HLS-GHCl lysis followed by Ampure XP, pick one concentration of SM59 and Nextera v1 with more replicates.

Goal: First, to extract, purify, and freeze six 1000 nuclei bulk DNA samples from 1-02 cerebellum sorted October 9, 2014. Second, create three libraries each with 1X SM59 (Stock is 5X) and 1:50 Nextera v1 and compare gels for intensity and distribution. Third, use SM59 to generate bulk libraries from 1-02 and 25-01 cortex with N = 3 for all.

Samples[edit]

To test SM59


sort # on tube nuclei/ul ul/tube nuclei/sample # samples # tubes to pool ul/sample ul HLS
1-02 cbl full 4 274 10 1000 6 3 3.65 31.35

Use 8-tube strip, including positive and negative control


Remaining bulk libraries


sort # on tube nuclei/ul ul/tube nuclei/sample # samples # tubes to pool ul/sample ul HLS
1-02 ctx full 1 120 3 1000 3 Not enough NA NA
1-02 ctx hi 2 120 3 1000 3 Not enough NA NA
1-02 ctx lo 3 78 10 1000 3 4 12.82 22.18
25-01 ctx full 5 283 20 1000 3 1 3.53 31.47
25-01 cbl full 6 232 10 1000 3 2 4.31 30.69

Protocol[edit]

Following protocol from January 22 up to Nextera

Lysis and freezedown[edit]

  1. Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
  2. 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
  3. Elute in 7 ul H2O
  4. Freeze at -20 C

Tagmentation[edit]

Tube 1 2 3 4 5 6 7 8
Sample 1-02 cbl 1-02 cbl 1-02 cbl 1-02 cbl 1-02 cbl 1-02 cbl NTC 6 ng gDNA
Condition 1:50 Nextera v1 1:50 Nextera v1 1:50 Nextera v1 1X SM59 1X SM59 1X SM59 1X SM59 1X SM59
  • Using SM59 on 5 tubes: 1-02 cerebellum 4-6, positive control, and NTC with supplied buffer.
    • Must mix A and B 1:1 --> 3 ul each
  • Using 1:50 Nextera v1 on 1-02 cerebellum 1-3 with in-house HMW buffer.
  • 5 minutes at 55 C
  • Standard ProtQ and Exo- treatments after tagmentation

PCR[edit]

  • Normal 2 step (6+7) PCR with indices 1, 2, and 5 for Nextera v1 tubes
  • 13 cycles with N5xx and N7xx for SM59 tubes