What happened to the U251_L3_T0_S19 and U251_L3_0GY_T0_S7_L001?

Are U343 RNA-Seq samples different from U251?

Are U343 Ribo-Seq samples different from U251?

Are U251 RNA-Seq T0 samples different from T1 and T24?

The L3_T0 RNA-Seq sample clusters with T24 samples.

Are U251 Ribo-Seq T0 samples different from T1 and T24?

The L3_T0 Ribo-Seq sample clusters with T24 samples similar to what happened at the RNA-Seq level.

How do we ensure that T0 is actually T24 and nothing else?

We first perform DE analysis by excluding ‘L3_T0’ from both RNA and Ribo-Seq. Let’s first concentrate at the RNA-Seq level. We pull out the list of the significantly downregulated genes between T0 and T24 in U251 RNA-seq. The expression values of these genes should be significantly different between the T24 vs T0 samples.

Are L1_T0 and L1_T24 different?

t.test(x = u251.rna.T24vsT0.normalized_counts$U251_L1_T0, u251.rna.T24vsT0.normalized_counts$U251_L1_T24)

    Welch Two Sample t-test

data:  u251.rna.T24vsT0.normalized_counts$U251_L1_T0 and u251.rna.T24vsT0.normalized_counts$U251_L1_T24
t = 3.3445, df = 2510.6, p-value = 0.0008365
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
  596.0444 2285.5745
sample estimates:
mean of x mean of y 
 6450.545  5009.735 

L1_T0 and L1_T24 are significantly different.

Are L1_T0 and L2_T0 different?

t.test(x = u251.rna.T24vsT0.normalized_counts$U251_L1_T0, u251.rna.T24vsT0.normalized_counts$U251_L2_T0)

    Welch Two Sample t-test

data:  u251.rna.T24vsT0.normalized_counts$U251_L1_T0 and u251.rna.T24vsT0.normalized_counts$U251_L2_T0
t = 0.038187, df = 2707.9, p-value = 0.9695
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
 -939.7513  977.0808
sample estimates:
mean of x mean of y 
 6450.545  6431.880 

No. As expected, they should be similar.

Are L1_T0 and L3_T0 different?

t.test(x = u251.rna.T24vsT0.normalized_counts$U251_L1_T0, u251.rna.T24vsT0.normalized_counts$U251_L3_T0)

    Welch Two Sample t-test

data:  u251.rna.T24vsT0.normalized_counts$U251_L1_T0 and u251.rna.T24vsT0.normalized_counts$U251_L3_T0
t = 3.8798, df = 2419, p-value = 0.0001073
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
  806.3778 2454.4907
sample estimates:
mean of x mean of y 
 6450.545  4820.111 

Yes! But they ought to be similar.

Are L3_T0 and L3_T24 different?

t.test(x = u251.rna.T24vsT0.normalized_counts$U251_L3_T0, u251.rna.T24vsT0.normalized_counts$U251_L3_T24)

    Welch Two Sample t-test

data:  u251.rna.T24vsT0.normalized_counts$U251_L3_T0 and u251.rna.T24vsT0.normalized_counts$U251_L3_T24
t = 0.091271, df = 2707.2, p-value = 0.9273
alternative hypothesis: true difference in means is not equal to 0
95 percent confidence interval:
 -641.2228  703.8306
sample estimates:
mean of x mean of y 
 4820.111  4788.807 

No! But they should be!

Ribo-Seq levels

Let’s do a similar analysis for the Ribo-Seq levels. This time we use up-regulated genes at the transaltion level only (T24vsT0.)