13. To DNase-treated RNA add 80 μL of RNase-free water.
14. Add 35 μL 8 M LiCl and precipitate RNA at 4
C overnight.
15. Microcentrifuge at 12,000 Â g at 4
C for 45 min and discard
supernatants.
16. Wash RNA pellets with ethanol prechilled at À20
C and
microcentrifuge at 12,000 Â g at 4
C for 5 min.
17. Repeat the washing step.
18. Air-dry RNA pellet.
3.7 Libraries
1. Resuspend RNA (see step 18 in Subheading 3.6) in 18 μL
Elute, Prime, Fragment Mix of the Illumina TruSeq
® sample
preparation Kit.
2. Libraries are generated following the Low sample
(LS) protocol of the Illumina TruSeq
® sample preparation kit
that omits the two rounds of poly(A) selection commonly used
for total RNA as a starting material.
3. For quality control, the size distribution is assessed using a
DNA-1000 Chip (Agilent) on an Agilent Technologies 2100
Bioanalyzer.
4. The concentration is determined using the Qubit dsDNA HS
assay kit according to the manufacturer’s protocol.
5. Sequencing of the libraries is performed using standard Illumina sequencers.
6. Bioinformatics to determine transcripts significantly enriched
in RBP precipitates were performed using standard tools to
determine differentially expressed genes, either transcripts significantly enriched to RNA-seq data of input controls as
described [7] or transcripts significantly enriched relative to
mock precipitations, e.g., performed with wild-type plants
lacking the RBP-GFP fusion [6].
4 Notes
1. Although we have not seen disadvantageous effects, VRC may
inhibit the RT reaction. In this case, VRC should be omitted or
depleted by adding 10 equivalents of EDTA before the isopropanol precipitation (see step 6 in Subheading 3.6).
2. Commercial versions of Tri-reagent are available, e.g., Trizol
(Invitrogen)
http://tools.invitrogen.com/content/sfs/
manuals/trizol_reagent.pdf.
3. Specificity and efficiency of formaldehyde cross-linking depend
on the duration of the treatment. We recommend performing a
pilot time-course for the RNA-binding protein under study.
RIP-seq
459
14. Add 35 μL 8 M LiCl and precipitate RNA at 4
C overnight.
15. Microcentrifuge at 12,000 Â g at 4
C for 45 min and discard
supernatants.
16. Wash RNA pellets with ethanol prechilled at À20
C and
microcentrifuge at 12,000 Â g at 4
C for 5 min.
17. Repeat the washing step.
18. Air-dry RNA pellet.
3.7 Libraries
1. Resuspend RNA (see step 18 in Subheading 3.6) in 18 μL
Elute, Prime, Fragment Mix of the Illumina TruSeq
® sample
preparation Kit.
2. Libraries are generated following the Low sample
(LS) protocol of the Illumina TruSeq
® sample preparation kit
that omits the two rounds of poly(A) selection commonly used
for total RNA as a starting material.
3. For quality control, the size distribution is assessed using a
DNA-1000 Chip (Agilent) on an Agilent Technologies 2100
Bioanalyzer.
4. The concentration is determined using the Qubit dsDNA HS
assay kit according to the manufacturer’s protocol.
5. Sequencing of the libraries is performed using standard Illumina sequencers.
6. Bioinformatics to determine transcripts significantly enriched
in RBP precipitates were performed using standard tools to
determine differentially expressed genes, either transcripts significantly enriched to RNA-seq data of input controls as
described [7] or transcripts significantly enriched relative to
mock precipitations, e.g., performed with wild-type plants
lacking the RBP-GFP fusion [6].
4 Notes
1. Although we have not seen disadvantageous effects, VRC may
inhibit the RT reaction. In this case, VRC should be omitted or
depleted by adding 10 equivalents of EDTA before the isopropanol precipitation (see step 6 in Subheading 3.6).
2. Commercial versions of Tri-reagent are available, e.g., Trizol
(Invitrogen)
http://tools.invitrogen.com/content/sfs/
manuals/trizol_reagent.pdf.
3. Specificity and efficiency of formaldehyde cross-linking depend
on the duration of the treatment. We recommend performing a
pilot time-course for the RNA-binding protein under study.
RIP-seq
459