2.4 Equipment
1. Mortar and pestle.
2. Teflon pestle and glass tube homogenizer.
3. Standard glass centrifuge tubes, 50 mL.
4. Standard microfuge tubes, 1.5 mL.
5. Standard thin-wall PCR tubes, 0.2 mL.
6. Low-retention microfuge tubes, 1.5 mL.
7. Standard lab centrifuges for microfuge tubes and conical tubes.
8. Neodymium magnet separation racks for 1.5 mL, 15 mL, and
50 mL (see Note 5).
9. Standard lab centrifuge for 50 mL tubes.
10. Standard thermocycler.
11. Back-and-forth shaker.
12. NanoDrop microvolume spectrophotometer.
13. Agilent 2100 BioAnalyzer.
14. RNA-SEQ data analysis: 64-bit computer with at least 1 Tb
hard disk space and 16 Gb of memory.
3 Methods
All steps in this protocol are carried out with solutions and materials
free of nucleases at 4
C. Commercial RNase/DNase-free pipette
tips and microcentrifuge tubes can be used without autoclaving.
Glassware, mortars, and pestles should be cleaned extensively and
must be sterilized by autoclaving. Miracloth must be autoclaved as
well. The protocol describes the procedure for plant tissue expressing a FLAG-tagged version of RPL18. For other organisms see
Table 1.
3.1 Tissue Collection
and Polysome
Extraction
1. Harvest tissue from transgenic plants expressing FLAG-RPL18
and flash-freeze in liquid nitrogen. Grind to a fine powder
maintaining the tissue frozen at all times (see Note 6).
2. Add approximately one volume of frozen packed pulverized
tissue to at least two volumes of PEB and mix with a glass bar
(see Note 7).
3. Let the mixture stand on ice while thawing.
4. Transfer the mixture to a glass homogenizer and make ten
strokes to homogenize the mixture.
5. Incubate the mixture on ice for 10 min.
6. Transfer the mixture to a 50 mL or a 15 mL centrifuge tube
and centrifuge at 16,000 Â g for 15 min at 4
C.
TRAP-SEQ Analysis of Long Noncoding RNAs
459
1. Mortar and pestle.
2. Teflon pestle and glass tube homogenizer.
3. Standard glass centrifuge tubes, 50 mL.
4. Standard microfuge tubes, 1.5 mL.
5. Standard thin-wall PCR tubes, 0.2 mL.
6. Low-retention microfuge tubes, 1.5 mL.
7. Standard lab centrifuges for microfuge tubes and conical tubes.
8. Neodymium magnet separation racks for 1.5 mL, 15 mL, and
50 mL (see Note 5).
9. Standard lab centrifuge for 50 mL tubes.
10. Standard thermocycler.
11. Back-and-forth shaker.
12. NanoDrop microvolume spectrophotometer.
13. Agilent 2100 BioAnalyzer.
14. RNA-SEQ data analysis: 64-bit computer with at least 1 Tb
hard disk space and 16 Gb of memory.
3 Methods
All steps in this protocol are carried out with solutions and materials
free of nucleases at 4
C. Commercial RNase/DNase-free pipette
tips and microcentrifuge tubes can be used without autoclaving.
Glassware, mortars, and pestles should be cleaned extensively and
must be sterilized by autoclaving. Miracloth must be autoclaved as
well. The protocol describes the procedure for plant tissue expressing a FLAG-tagged version of RPL18. For other organisms see
Table 1.
3.1 Tissue Collection
and Polysome
Extraction
1. Harvest tissue from transgenic plants expressing FLAG-RPL18
and flash-freeze in liquid nitrogen. Grind to a fine powder
maintaining the tissue frozen at all times (see Note 6).
2. Add approximately one volume of frozen packed pulverized
tissue to at least two volumes of PEB and mix with a glass bar
(see Note 7).
3. Let the mixture stand on ice while thawing.
4. Transfer the mixture to a glass homogenizer and make ten
strokes to homogenize the mixture.
5. Incubate the mixture on ice for 10 min.
6. Transfer the mixture to a 50 mL or a 15 mL centrifuge tube
and centrifuge at 16,000 Â g for 15 min at 4
C.
TRAP-SEQ Analysis of Long Noncoding RNAs
459