LaCava Research Wiki

AIM/HYPOTHESIS

See Endogenous a-ORF1 RNA-seq_Lars.

The idea of this test experiment is to quickly test our methodology. Mainly, to establish how much powder we need per IP to capture enough RNA for proper sequencing data. Furthermore, we check the specificity of the RNA binding. To achieve this I will do a-ORF1 IPs on different amounts of powder of both N2102Ep and NTERA2 (due to abundance of material), take along mouse IgG controls, have the appropriate samples quickly sequenced at the genomics facility here at RU to check the data quality and then move to prepping the RNA of the samples mentioned in Endogenous a-ORF1 RNA-seq_Lars.

MATERIALS

• Nuclease free extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor); RNAsin 1:250 in extraction buffer, 1:1000 in wash buffer.
• Scale: 50 or 200mg cell powder per IP reaction with 10ul of beads per 50mg

METHODS

All buffers made with RNAse-free H₂0.

Samples (+sonication energy output)

1)	N2102Ep 50mg a-ORF1 (10µl beads) | 11J
2)     N2102Ep 50mg a-mIgG (10µl beads) | 12J
3)	N2102Ep 200mg a-ORF1 (40µl beads) | 11+9+9+10=39J
4)	NTERA 50mg a-ORF1 (10µl beads) | 11J
5)	NTERA 50mg a-mIgG (10µl beads) | 9J
6)	NTERA 200mg a-ORF1 (40µl beads) | 10+9+10+12=41J

Experiment was performed as stated in RNA extraction after cell powder IP

RESULTS

The sample numbers correspond to the number above.

Our yields were:

1)	N2102Ep 50mg a-ORF1 | 18,872 pg/μl
2)	N2102Ep 50mg a-mIgG | 328 pg/μl
3)	N2102Ep 200mg a-ORF1 | 33,038 pg/μl
4)	NTERA 50mg a-ORF1 | 9,570 pg/ul
5)	NTERA 50mg a-mIgG | 99 pg/ul
6)	NTERA 200mg a-ORF1 | 27,083 pg/μl

DISCUSSION

The first thing that we can conclude from these yields is that the RNA is definitely ORF1 specific, so we can in fact deduce the L1 RNA yield from rRNA levels.

More important, 50mg scale seems to be sufficient for RNA-seq. I compared the RNA yield of this experiment to the amounts that were used in the eLife paper (https://doi.org/10.7554/eLife.30094.001), which provided proper data. Elutions there were done with 14uL, compared to 6 now. Losing 1uL when eluting and 1 for bioanalyzer analysis leaves us with ~12 vs. ~4uL of RNA sample. As can be seen here 10/16/17 - ORF2 (LD567 and LD624) anti-FLAG IP for RNA-seq > RNA Samples for Library_101717, previous yield ranged from ~1,000 to 9,000 pg/uL and was sufficient for good RNA-seq data. This means that we need at least a yield of 3,000 pg/uL sample to match this and preferably higher.

To be on the save side, and given that we have enough material, I propose we go for 100mg scale to prepare the RNA samples.