2. Add 800 μL of TRIzol to each sample, vortex for 30 s, and
incubate at room temperature for 5 min to allow the dissociation of ribonucleoprotein complexes.
3. Add 200 μL of chloroform, shake the tube vigorously by hand
for 15 s, and incubate at room temperature for 3 min.
4. Centrifuge at 12,000 Â g for 15 min at 4
C.
5. Transfer the clear upper phase to a new 1.5 mL microfuge tube.
6. Repeat the chloroform extraction steps 3–5 to remove residual
TRIzol traces that can inhibit downstream enzymatic reactions.
7. To precipitate RNA, add 500 μL of isopropanol, invert the
tube gently by hand to mix, and incubate at room temperature
for 10 min or at À20
C overnight. For samples with low yields
of affinity-purified polysomes (less than 100 ng), add 1 μL of
20 μg/μL glycogen along with the isopropanol, to aid in the
precipitation.
8. Centrifuge at 12,000 Â g for 15 min at 4
C. The RNA pellet is
visible after centrifugation. Discard the supernatant as hazardous waste following local regulations.
9. Wash pellet with 1 mL of 70% (v/v) ethanol. Centrifuge at
7,500 Â g for 5 min at 4
C.
10. Discard supernatant and let the pellet air-dry at room temperature for 20 min.
11. Resuspend the pellet in 50 μL of molecular biology-grade
water by incubating the sample at 60
C for 10 min to allow
the complete resuspension of the RNA pellet. Store at À80
C
until use.
12. Use 1 μL of each sample to estimate the yield and concentration of RNA with a NanoDrop spectrophotometer according
to the manufacturer’s instructions (see Note 10).
13. Evaluate the quality of the sample using an Agilent 2100
Bioanalyzer with RNA 6000 Nano or Pico Assay reagent kits
(see Note 11).
3.5 Preparation
and Sequencing
of RNAseq Illumina
Compatible Libraries
1. Use an RNA-seq kit of choice and follow the manufacturer’s
protocol or the homemade version available at your lab (see
Note 12).
2. For DNA purification steps use AMPure XP Beads. The range
of DNA fragments to purify can be manipulated by changing
the concentration of PEG in the buffer (Fig. 4; see Note 13).
3. For indexing of the libraries choose a manufacturerrecommended combination of adapters to allow multiplex
sequencing. The procedures can take 1–2 days.
462
Soledad Traubenik et al.
incubate at room temperature for 5 min to allow the dissociation of ribonucleoprotein complexes.
3. Add 200 μL of chloroform, shake the tube vigorously by hand
for 15 s, and incubate at room temperature for 3 min.
4. Centrifuge at 12,000 Â g for 15 min at 4
C.
5. Transfer the clear upper phase to a new 1.5 mL microfuge tube.
6. Repeat the chloroform extraction steps 3–5 to remove residual
TRIzol traces that can inhibit downstream enzymatic reactions.
7. To precipitate RNA, add 500 μL of isopropanol, invert the
tube gently by hand to mix, and incubate at room temperature
for 10 min or at À20
C overnight. For samples with low yields
of affinity-purified polysomes (less than 100 ng), add 1 μL of
20 μg/μL glycogen along with the isopropanol, to aid in the
precipitation.
8. Centrifuge at 12,000 Â g for 15 min at 4
C. The RNA pellet is
visible after centrifugation. Discard the supernatant as hazardous waste following local regulations.
9. Wash pellet with 1 mL of 70% (v/v) ethanol. Centrifuge at
7,500 Â g for 5 min at 4
C.
10. Discard supernatant and let the pellet air-dry at room temperature for 20 min.
11. Resuspend the pellet in 50 μL of molecular biology-grade
water by incubating the sample at 60
C for 10 min to allow
the complete resuspension of the RNA pellet. Store at À80
C
until use.
12. Use 1 μL of each sample to estimate the yield and concentration of RNA with a NanoDrop spectrophotometer according
to the manufacturer’s instructions (see Note 10).
13. Evaluate the quality of the sample using an Agilent 2100
Bioanalyzer with RNA 6000 Nano or Pico Assay reagent kits
(see Note 11).
3.5 Preparation
and Sequencing
of RNAseq Illumina
Compatible Libraries
1. Use an RNA-seq kit of choice and follow the manufacturer’s
protocol or the homemade version available at your lab (see
Note 12).
2. For DNA purification steps use AMPure XP Beads. The range
of DNA fragments to purify can be manipulated by changing
the concentration of PEG in the buffer (Fig. 4; see Note 13).
3. For indexing of the libraries choose a manufacturerrecommended combination of adapters to allow multiplex
sequencing. The procedures can take 1–2 days.
462
Soledad Traubenik et al.