Arichard:Notebook/bulk150210
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]
- Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
- 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
- Elute in 7 ul H2O
- 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