# Notes on RNA progress for thermal block experiments

### Strain 2090

Position 3 (low temp) already has high-quality RNA extraction + all nutrient filters done.

| ID on Bioanalyzer | Material Submitted | ID Submitted | Bioanalyzer Concentration (ng/uL) | Dilution Amount added to 1 uL sample | Bioanalyzer Adjusted by Dilution (ng/uL) | RIN | Concentration Submitted Determined By | Concentration Submitted (ng/uL) | Total volume Submitted (uL) | Total Submitted (ng) | Qubit | Descriptive |
|:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |
| 1 Arianna (1/13 15:33) | Total RNA | HA057 | 2.804 | 0 | 2.804 | N/A | Bioanalyzer | 2.804 | 28 | 78.512 | | Strain 2090 LOW R1 |
| 2 Arianna (1/13 15:33) | Total RNA | HA058 | 2.536 | 0 | 2.536 | N/A | Bioanalyzer | 2.536 | 28 | 71.008 | | Strain 2090 LOW R2 |
| 3 (1/13 15:00) | Total RNA | HA059 | 6.279 | 0 | 6.279 | N/A | Bioanalyzer | 6.279 | 25 | 156.975 | | Strain 2090 LOW R3 |

Positions 13 & 19 have 2 extractions of high quality and 1 of low quality.

| ID on Bioanalyzer | Material Submitted | ID Submitted | Bioanalyzer Concentration (ng/uL) | Dilution Amount added to 1 uL sample | Bioanalyzer Adjusted by Dilution (ng/uL) | RIN | Concentration Submitted Determined By | Concentration Submitted (ng/uL) | Total volume Submitted (uL) | Total Submitted (ng) | Qubit | Descriptive |
|:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |:---: |
| 13A (1/18) | Total RNA | HA060 | 2.944 | 0 | 2.944 | N/A | Bioanalyzer | 2.944 | 28 | 82.432 | | Strain 2090 MID R1 |
| 13B (1/18) | Total RNA | HA061 | 2.666 | 0 | 2.666 | N/A | Bioanalyzer | 2.666 | 28 | 74.648 | | Strain 2090 MID R2 |
| 13C (1/18) | Total RNA | HA062 | 1.379 | 0 | 1.379 | N/A | Bioanalyzer | 1.379 | 28 | 38.612 | | Strain 2090 MID R3 |
| 19A (1/18) | Total RNA | HA063 | 2.609 | 0 | 2.609 | N/A | Bioanalyzer | 2.609 | 28 | 73.052 | | Strain 2090 HIGH R1 |
| 19B (1/18) | Total RNA | HA064 | 1.09 | 0 | 0.954 | 4.6 | Bioanalyzer | 1.09 | 29 | 31.61 | | Strain 2090 HIGH R2 |
| 19C (1/18) | Total RNA | HA065 | 2.092 | 0 | 2.092 | 5.6 | Bioanalyzer | 2.09 | 29 | 60.61 | | Strain 2090 HIGH R3 |

I retrieved additional nutrient filters for these early this week and later this week will get new RNA filters and extract.

### Strain 1516

Strain 1516 has been more challenging to pinpoint definitively w.r.t. thermal optimum. I was worried that position 16 might be past optimum so was collecting 12 instead, but now it appears as though position 16 is the right choice. For both 2090 and 1516, as the experiment has progressed, I have had less of a "past-optimum".


The cell size data is also inconclusive for both 1516 and 2090, which supports my previous observations in my past experiments w/ the uncalcified.


1516 and 2090 began quite differentiated from one another w.r.t. both cell size and redB fluorescence. Now they are hardly distinguishable w.r.t. size in the L1/25 media (probably the culprit), but remain differentiated w.r.t. centering of population redB.


With regard to media, media appears to increase GR of 2090, which would be the opposite of the trend I saw with the change in 2090 cell size. But I'll have to check this out in more detail, as Suzana also asked about the shift in cell size distribution which might happen w/ a change in media.


We could easily add a few tests for 1516 and 2090 since their target temperatures are easy to replicate ex-thermal-block.

As far as 1516 RNA, I have a similar situation: 2 of 3 replicates are high-quality and not degraded for all 3 temperatures. I have new filters for position 6 and expect to collect new filters for 16 and 20 later this week. We have been conducting some testing with the input lysis buffer for the extraction protocol. It didn't help as much as I would have liked to split into 2 filters + 2 bead tubes. In the next round of tests I will simply get the filters into smaller pieces and add additional lysis buffer.