Records

From Neurobiology.Dev

Below is a list of all experimental records and data. Records typically include a sample ID as well as a link to files for that run. These files are stored on a google drive that will require access in order to download. Some of these files have been compressed to reduce file size and increase the ease of transferring to and from storage. All records here are backed up on the lab drive in their respective project folders as well.

Tip

I have begun to compress these files for long term storage in the cloud and on the lab drive. They can be compressed with the following terminal command (Mac) tar -czf foldername.tar.gz foldername. These can be uncompressed with tar –xf foldername.tar.gz.

It is also noteworthy that you can download from the google drive in R directly using library("googledrive") and drive_download("filename.tar.gz", path = "outputpath/filename.tar.gz", overwrite=TRUE). If no input path is given and you just put "filename.tar.gz" in both fields, it will download to R's current working directory.

M6A G-RAMP

Below is a sortable table for all Nanopore m6A RNA runs conducted in the Lai Lab. These data are also stored on the lab drive in addition to a google drive backup.

Entry Sample ID (Notebook Reference) Reports(s) / Data Protocol Cell Line IDH Status Output Reads Notes Date of Run
29 FA45LB5121MUT524 (LLT-001p131)

Nanopore RNA Sequencing Protocol LB5121 MUT - - Nanopore run using RNA flow cell 5/21/24
28 FA38LB5139WT524 (LLT-001p131)

Nanopore RNA Sequencing Protocol LB5139 WT Good 2.08M First nanopore run with a patient sample using the new RNA flow cells. Spiked in C. Luciferase 300 base RNA ligated to 100 base m6A positive and negative controls, but it didn't come through. 5/17/24
27 AS76LB4766WT324 (LLT-001p108)

Nanopore RNA Sequencing Protocol LB4766 WT Okay 22.69k Same sample as previous entry, used remaining library amount. 59 pore flow cell. 3/25/24
26 AS56LB4766WT324 (LLT-001p108)

Nanopore RNA Sequencing Protocol LB4766 WT Good 110.81k Same sample as previous entry, but I loaded only 5 μL of eluted library since library was very concentrated. Good results. 90-100 pore flow cell. 3/22/24
25 AS47LB4766WT324 (LLT-001p108)

Nanopore RNA Sequencing Protocol LB4766 WT Good 70.31k Same sample as previous entry, but I loaded only 5 μL of eluted library since library was very concentrated. Good results. 73 pore flow cell. 3/21/24
24 AS32LB4766WT324 (LLT-001p107)

Nanopore RNA Sequencing Protocol LB4766 WT Okay 38.19k Extraction and run with LB4766 sample. mRNA from this sample appeared dilute (21.3 ng/μL), so I used 10 μL mRNA for nanopore library prep, reducing H20 to 8 μL in step 5. Eluted in 15 μL of buffer at the end of the Nanopore protocol. 7 μL of final library sample was loaded with 9 μL / 14 μL LBII and SBII. Flow cell appeared overloaded. 3/20/24
23 AS80LB4424MUT324 (LLT-001p106)

Nanopore RNA Sequencing Protocol LB4424 Mut Good 64.19k Library prep using frozen mRNA extracted from LB4424. Concentration of the mRNA input was low (19 ng/μL), so I increased the input slightly and decreased the water loaded at step 5 in the protocol. Loaded 10 μL of library with 9 μL / 14 μL LBII and SBII. Might have slightly overloaded the flow cell. 3/18/24
22 AW25LB5121MUT324 (LLT-001p105)

Nanopore RNA Sequencing Protocol LB5121 Mut Good 82.89k Left over library run a day later from the previous protocol. Ended up loading ~10 μL of library (remaining) and 9 μL / 14 μL of loading beads and sequencing buffer. Note: Naming for this one was incorrect for the flow cell ID, because I was in a hurry. I left it as is so it matches with the folder in minKNOW on the linux workstation. 3/15/24
21 AS52LB5121MUT324 (LLT-001p105)

Nanopore RNA Sequencing Protocol LB5121 Mut Good 84.81k Run on an IDH-Mut patient sample. Extracted total and mRNA on the same day as the run using the direct mRNA kit and TRIzol. Note: Naming for this one was incorrect for the flow cell ID, because I was in a hurry. I left it as is so it matches with the folder in minKNOW on the linux workstation. 3/13/24
20 AQ14LB4424MUT224 (LLT-001p101)

Nanopore RNA Sequencing Protocol LB4424 Mut Poor 4.41k Reused the same library from the previous day, but on a new Flongle. Flongle was below warranty and the re-used library was suboptimal after freeze/thaw. 2/22/24
19 AQ86LB4424MUT224 (LLT-001p101)

Nanopore RNA Sequencing Protocol LB4424 Mut Okay 32.01k This was on a sub-optimal below warranty Flongle (36 pores available). This run was different because I re-measured the concentration of mRNA after thawing, loading 9 μL of 35 ng/μL starting mRNA. Eluted the final elute volume into 15 μL instead of 21 μL, and loaded 7 μL library (with 9 μL LBII and 14 μL SB). The library prep was good and had good pore occupancy, but the Flongle was a below warranty Flongle. 2/21/24
18 AQ84LB4424MUT224 (LLT-001p100)

Nanopore RNA Sequencing Protocol LB4424 Mut Okay 22.44k Run using left over library from the previous two runs. This was on a sub-optimal below warranty Flongle. 2/20/24
17 AQ23LB4424MUT224 (LLT-001p100)

Nanopore RNA Sequencing Protocol LB4424 Mut Okay 23.95k Run using left over library from the previous run. This was on a sub-optimal below warranty Flongle. 2/17/24
16 AQ12LB4424MUT224 (LLT-001p100)

Nanopore RNA Sequencing Protocol LB4424 Mut Okay 39.87k Run using patient mRNA that had been previously frozen and stored at -80ºC. Eluted into 21 μL twice. Used 7 μL library, 9 μL LBII and 14 μL SB. This was done on a Flongle with 66 pores available, but note that this Flongle had previously reported 71 pores (8/1/23) and 1 pore (1/25/24) respectively. Flongle slightly underloaded, which I found out later after having to pipette mRNA after freezing which showed a lower concentration. 2/16/24
15 AQ73LB5139WT124 (LLT-001p96)

Nanopore RNA Sequencing Protocol LB5139 WT Okay 32.23k Run using left over prepared library from the previous two entries, frozen at -20ºC and thawed twice. The flow cell was below warranty. Pore decay and pore occupancy were optimal for a below warranty flow cell. 1/27/24
14 AQ61LB5139WT124 (LLT-001p96)

Nanopore RNA Sequencing Protocol LB5139 WT Okay 45.42k Run using left over prepared library from the previous entry, frozen at -20ºC and thawed the next day. The flow cell used was one near below warranty (~52 pores available at the start). Pore decay and pore occupancy were optimal. 1/24/24
13 AQ97LB5139WT124 (LLT-001p96)

Nanopore RNA Sequencing Protocol LB5139 WT Good 102.68k First run with patient tissue. Patient was a GBM grade IV. Starting material was a 60 mg piece of tumor that was run through a trizol total RNA extraction protocol, followed by mRNA purification using the direct mRNA kit. RNA was exceptionally pure. 1/23/24
12 AP56HK217W124 (LLT-001p91)

Nanopore RNA Sequencing Protocol HK217 WT Okay 44.38k Used the mRNA extracted from the previous run, so it was a few days old and had been freeze/thawed. I spiked in m6A positive and negative controls (0.5 μL each) and 9 μL of the mRNA extract that was around 225 ng total. Pore decay was a tad faster; I eluted in 13 μL at the end to try to improve throughput from entry 11. No real improvement. Input mRNA should be 330 ng always. 1/11/24
11 AQ43HK217W124 (LLT-001p90)

Nanopore RNA Sequencing Protocol HK217 WT Okay 42.19k This was freshly extracted mRNA, no rRNA cleanup step. Input mRNA was ~25 ng/μL, for about 225 ng total loaded in 9 μL. Pore decay was slow, the Flongle was just underloaded. 1/10/24
10 AQ45U8W1123 (LLT-001p87)

Nanopore RNA Sequencing Protocol U87 WT Good 81.02k This was freshly extracted mRNA, no rRNA cleanup step. Differences in this run: mRNA input was checked for 2.0 or greater ratio in 260/230 and 260/280 regions. mRNA input was the full 9 μL, for around 333 ng of mRNA total. I also turned the pellet fast during ethanol wash, pipetted instead of flicked the tube, and eluted twice. These changes were added to the protocol. 11/09/23
9 FC14U8W723 (LLT-001p71-72)

Nanopore RNA Sequencing Protocol U87 WT Good 56k This was a test run using stored U87 cell RNA that had been purified for mRNA prior to the run. This run generated a decent output. 07/14/23
8 FC31HKW623 (LLT-001p67)

Nanopore RNA Sequencing Protocol HK217 WT Okay - This was a control run, which mostly contained synthetic RNA oligos containing Gluc/Cluc and m6A modifications. Run on a flongle flow cell. HK217 IDH-WT spiked in as a control. 06/14/23
7 FC92HKW623 (LLT-001p63)

Nanopore RNA Sequencing Protocol HK217 WT Poor - This was the first attempt at a control run, which mostly contained synthetic RNA oligos containing Gluc/Cluc and m6A modifications. Run on a flongle flow cell. HK217 IDH-WT spiked in as a control. 06/02/23
6 FC04HKW323 (LLT-001p24-25)

Nanopore RNA Sequencing Protocol HK217 WT Good 57.41k This run was a decent run using a Flongle Flow Cell and a gliomasphere cell line. 03/16/23
5 FR56U8W1221 Nanopore RNA Sequencing Protocol U87 WT Good - This was the vector control U87 re-run by Terry Prins and Sean Pianka. This run yielded a good throughput. 12/20/21
4 FQ15U8W921 Nanopore RNA Sequencing Protocol U87 WT Poor - This is a vector control U87 sample run by Bryan Kevan and Sean Pianka. This run yielded a very low amount of reads before failing. 09/17/21
3 FO96U8W521 Nanopore RNA Sequencing Protocol U87 WT Poor - This is a vector control U87 sample run by Bryan Kevan and Sean Pianka. This run did not yield an optimal amount of reads. 05/05/21
2 FM31U8M720 Nanopore RNA Sequencing Protocol U87 Mut Good - This sample was a U87 sample run by Bryan Kevan and Sean Pianka. 07/15/20
1 FM68U8W720 Nanopore RNA Sequencing Protocol U87 WT Poor - This is a vector control U87 sample run by Bryan Kevan and Sean Pianka. This run did not yield an optimal amount of reads. 07/14/20