A | B | |
5’ adapter | /5AzideN/GTTCAGAGTTCTACAGTCCGACGATC[CTGATC]rNrNrNrNrNrNrN | |
3’ adapter | /5rApp/NN NNT GGA ATT CTC GGG TGC CAA GGA AAA AAA AAA AA/iAzideN/ AAA AAA AAA AAA /3Bio/ | |
small RNA RT Primer | GCCTTGGCACCCGAGAATTCCA | |
small RNA PCR F Primer | AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA | |
small RNA PCR R Primer I1 | CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I2 | CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I3 | CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I4 | CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I5 | CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I6 | CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I7 | CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA | |
small RNA PCR R Primer I8 | CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCCTTGGCACCCGAGAATTCCA |
final concentration | for 500 ml | |
50 mM Tris–HCl, pH 7.4 | 25 ml of 1M | |
100 mM NaCl | 10 ml of 5M | |
1% Igepal CA-630 | 5 ml of 100% | |
0.1% SDS | 2.5 ml of 20% | |
0.5% sodium deoxycholate | 25 ml of 10% |
final concentration | for 250 ml | |
50 mM Tris–HCl, pH 7.4 | 12.5 ml of 1M | |
1 M NaCl | 50 ml of 5M | |
1 mM EDTA | 0.5 ml of 0.5M | |
1% Igepal CA-630 | 2.5 ml of 100% | |
0.1% SDS | 1.25 ml of 20% | |
0.5% sodium deoxycholate | 12.5 ml of 10% |
final concentration | for 10 ml | for 500 ml | |
20 mM Tris–HCl, pH 7.4 | 0.2 ml of 1M | 10 ml | |
10 mM MgCl 2 | 0.1 ml of 1M | 5 ml | |
0.2% Tween-20 | 20 µl of 100% | 1 ml |
final concentration | for 100 µl of 5x buffer | |
350 mM Tris–HCl, pH 6.5 | 35ul of 1M | |
50 mM MgCl2 | 5ul of 1M | |
5 mM dithiothreitol | always add fresh 1 µl of 1M per 200ul 1x reaction |
final concentration | for 10 ml | |
0.1M Tris-HCl, pH 7.5 | 1 ml of 1M | |
1M NaCl | 2 ml of 5M | |
0.1% Tween-20 | 10 µl of 100% | |
1mM EDTA | 20 µl of 0.5M |
final concentration | for 10 ml | |
50 mM Tris-HCl, pH 7.5 | 500 µl of 1M | |
150 mM NaCl | 300 µl of 5M | |
1 mM MgCl2 | 10 µl of 1M | |
0.5% NP-40 | 5 µl of 10% |
final concentration | for 10 ml | |
100 mM TrisCl pH 7.5 | 1 ml of 1M | |
50 mM NaCl | 100 µl of 5M | |
1 mM EDTA | 20 µl of 0.5M | |
0.2% SDS | 100 µl of 20% |
A | B | |
Dye in PBS | 20 µl | |
100µM oligo | 86 µl | |
PBS | to 200 µl (94 µl) |
Component | amount, µl for 20 µl reaction | |
Water, RNase-free | 15 | |
5x pH6.5 buffer | 4 | |
SuperasIn | 0.5 | |
T4 PNK | 0.5 | |
fresh DTT | to 5 mM |
Component | amount, µl for 20 µl reaction | |
Rnase-free water | 11 | |
3' adapter, labeled, from step 4 (diluted to 1 pmol/µl) | 1 | |
PEG8000 50% | 4 | |
10x T4 RNA ligase buffer | 2 | |
Rnl2TrK227Q | 1 | |
SuperaseIn | 1 | |
fresh DTT | to 5 mM |
Component | amount, µl for a 20 µl reaction | |
T4 PNK | 2 | |
SuperasIn | 1 | |
Rnase-free water | 13 | |
10x PNK buffer | 2 | |
10mM ATP | 2 | |
fresh DTT | to 5 mM |
Component | amount, µl for 20 µl reaction | |
T4 Rnl1 buffer 10x | 2 | |
PEG8000, 50% | 10 | |
10mM ATP | 2 | |
5' adapter from step 4 (diluted to 1 pmol/µl) | 1 | |
SuperaseIn | 0.5 | |
T4 RNA ligase 1 (normal or high concentration) | 1 | |
Rnase-free water | 4 | |
fresh DTT | to 5 mM |
component | amount, µl per 1 reaction | |
5x Superscript buffer | 4 | |
10 mM dNTPs | 1 | |
0.1M DTT | 1 | |
SuperaseIn | 0.5 | |
Superscript III | 0.5 | |
Rnase-free water | 1 |
Component | amount for 5 reactions, µl | |
cDNA | 5 | |
small RNA forward primer | 1 | |
small RNA reverse barcode (index) primer | 1 | |
NEBNext PCR MM | 25 | |
water | 18 |
component | amount, µl | |
rest cDNA | 15-17 | |
NEBNext 2x PCR MM | 25 | |
small RNA forward primer | 1 | |
small RNA reverse barcode (index) primer | 1 | |
water | to 50 |
Problem | Solution | |
No double ligated band is seen on the membrane after transfer | Troubleshoot possible steps at which material loss could have occurred (see below) | |
1. Not enough material | Try to increase the amount of input lysate at least 10-fold. Try to overexpress the RBP. | |
2. Immunoprecipitation is not working | Collect input, supernatant in IP samples in protein buffer for Western blot assessment of immunoprecipitation efficiency. Note that crosslniking rate of RBPs on average is only ~1%, so do not expect to see a Western blot signal where infrared ligated complexes are. If IP is not efficient, try a differnet antibody or tag the RBP of interest endogenously. FLAG tag usually works well. | |
2a. RBP is not in the soluble fraction | If you cannot detect your RBP in the input, try checking by Western blot the insoluble pellet after lysate centrifugation at step 7.2. Possibly you may need to change lysis buffer or add sonication if your RBP is very chromatin-bound. | |
3. Transfer is not working | Image the gel before and after transfer to the membrane. If the double-ligated band is still seen in the gel after transfer, try to increase transfer efficiency by reducing methanol, increasing SDS in transfer buffer and transferring for longer time. Try to increase RNase concentration to get shorter fragments. Alternatively, you can try the original PAR-CLIP protocol by cutting the band directly out of the gel and electroeluting the crosslinked complexes directly out of the gel piece. | |
4. Only adapter dimer and 3' end ligation product is seen on the gel, but not 5' or double ligation product | DTT in ligation buffer deteriorated. This can happen even you aliquot fresh NEB buffer and use aliquots only once. Always add fresh DTT diluted from 1M stock to 5 mM. | |
No library amplification in pilot PCR | ||
1. Not enough material | Extend pilot PCR up to 35-40 cycles. If the band of correct size is detected, try to increase the amount of input lysate at least 10-fold. | |
2. Not efficient Proteinase K digestion | Do use SDS-containing Proteinase K buffer. I had problems with digestion step when using urea-containing buffer. | |
3. Oligo-dT purification is not working | Check supernatants from the oligo-dT bead purification on an RNA urea-PAGE gel. | |
4. Reverse transcription is not working | Check your RT enzyme by performing an independent RT reaction with a positive control. | |
5. PCR is not working | Include a positive control to check the PCR master mix. Use the same primers for pilot PCR as for the main PCR. Check that your primers are from the correct kit (Illumina small RNA kit). (No joke: in case of NEXTflex kits, both small RNA and mRNA forward primers are called simply "Universal forward primer", and if it once gets into the wrong box, you will get no amplification). |