SAGE-Seq
(SAGE adapted for Solexa 1G platform)
This protocol is optimized for 5x10^4-2x10^6 cells, 0.5-50 μg total RNA, or 5-250 ng mRNA.
I. cDNA synthesis
Note:
There are many mixing and washing steps of the magnetic beads throughout this protocol. Mix the beads by flicking or gentle vortex. Centrifuge briefly at low speed to collect beads stuck on the tube cap. Do washes by pipetting the beads up and down several times, esp. when clumping occurs. If beads stick to the sides of tubes, scrape with pipette tip to recover them. In all the washing steps, add solution to the tube while it's still on the magnetic stand in order to minimize drying of the beads, then close the cap, remove it from the magnet and resuspend beads. When removing supernatant, place the pipette tip at the opposite side of the tube, away from beads, slide the pipette tip to the bottom and pipette up very slowly, so you won't disturb and lose the beads.
1. Thoroughly resuspend the oligo(dT) beads, transfer 100 ml to a RNase-free 1.5-ml siliconized tube and place on magnet. After 30 sec remove supernatant.
2. Resuspend beads in 500 ml lysis/bindig buffer by gentle vortex. Leave beads in this buffer until ready to mix them with the cell lysate/RNA sample, remove it right before that.
3. If using cells as starting material, lyse cells in 1 ml of lysis/binding buffer, reduce viscosity by pressing it through a 21 G needle using a 1ml syringe. If using total RNA or mRNA, adjust the volume to 1 ml with lysis/binding buffer. Add lysate/RNA to pre-washed oligo(dT) beads and incubate at RT for 5 min with constant agitation (by hand).
4. Place the tube on magnet for 2 min, remove supernatant (the supernatant from cell lysate can be saved and used for DNA prep.).
5. Wash 2x 1 ml of wash buffer A, 1x 1 ml of wash buffer B, 4x 100 ml of 1x 1st strand buffer (dilute 5x 1st strand buffer with DEPC treated water).
6. Resuspend beads in 1st strand synthesis mix:
DEPC water 54.5 ml
5x 1st strand buffer 18 ml
0.1 M DTT 9 ml
10 mM dNTP 4.5 ml
RNaseOUT 1 ml
87 ml
7. Place the tube at 37oC for 2 min, then add 3 ml of SuperScript RT and mix. Incubate at 37oC for 1 hr, mix beads every 10 min by hand vortexing. After incubation place tube on ice to terminate the reaction.
8. On ice, add the components of the 2nd strand synthesis, in the order shown, to the first strand reaction:
1st strand reaction 90 ml
DEPC water (prechilled) 477 ml
5x 2nd strand buffer 150 ml
10 mM dNTP 15 ml
E. coli DNA ligase 3 ml
E. coli DNA polymerase 12 ml
E. coli Rnase H 3 ml
750 ml
Mix and incubate at 16oC for 2 hours, mix beads every 10 min.
9. After incubation place tubes on ice and terminate reaction by adding 45 ml of 0.5 M EDTA. Place the tube on magnet for 2 min, remove supernatant.
10. Preheat wash buffer C to 75C. Wash 1x 200 ml of wash buffer C, resuspend beads in 200 ml wash buffer C and heat at 75C for 20 min to inactive E. coli DNA polymerase.
11. Wash 4x 200 ml of wash buffer D + BSA. Take 2.5 ml of the last wash of wash buffer D to check the integrity of cDNA by RT-PCR [for 2x 25 ml reactions of β-actin & the other gene. Thermal cycle: 94C, 3 min; 94C, 15 sec, 64oC, 30 sec, 72C, 1 min/kb (3 cycels); 94C, 15 sec, 61C, 30 sec, 72C, 1 min/kb (3 cycels); 94oC, 15 sec, 58oC, 30 sec, 72C, 1 min/kb (35 cycels); 72oC, 5 min].
12. Resuspend beads in 200 ml of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of 1x buffer 4 + BSA, combine the content. Wash once more with 200 ml of 1x buffer 4 + BSA. The beads can be stored in 1x buffer 4 + BSA at 4oC O/N.
Note:
Use 2x BSA in all the washing buffers (20 ml 100x/ml). More BSA seems to reduce stickiness and improves the efficiency of the washes and the quality of the library. After the wash buffer C washing/heating step the beads are stickier until the first BSA wash, but then they are OK.
---------------------------------------------PAUSE POINTS---------------------------------------------------
II. Cleavage of cDNA with anchoring enzyme (NlaIII)
1. Resuspend beads in following mix and incubate at 37oC for 1 hour. Mix beads every 10 min.
dH2O 170 ml
100x BSA 4 ml
10x buffer 4 20 ml
NlaIII 6 ml
200 ml
2. Place the tube on magnet for 1 min, remove supernatant. Preheat wash buffer C to 37oC to prevent precipitation of SDS. Wash 2x 200 ml of wash buffer C to inactivate NlaIII.
3. Wash 1x 200 ml wash buffer D + BSA. Resuspend beads in 200 ml of wash buffer D + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of wash buffer D + BSA, combine the content. Wash 2x 200 ml wash buffer D + BSA. The beads can be very clumpy at this point, so be patient and keep pipetting them up and down until they are not clumpy.
4. Wash 2x 200 ml 1x ligase buffer. At final rinse transfer to a new siliconized tube.
III. Ligating Gex Adapter 1 to cDNA
Gex Adapter 1:
5'-ACAGGTTCAGAGTTCTACAGTCCGACATG-3’
3’-CAAGTCTCAAGATGTCAGGCT-phos-5’
1. Remove last wash and resuspend beads as follows:
dH2O 6 ml
5x ligase buffer 2 ml
Gex Adapter 1 (10 μM 1* ml
9 ml
*The amount of adapters used needs to be adjusted according to the number of cells.
2. Heat tubes at 50oC for 2 min. Let sit at RT for 10 min. Add 1 ml of T4 ligase (high conc.) to each tube and incubate at 16oC for 2 hours. Mix beads every 15 min.
3. Preheat wash buffer C to 37oC. Wash 2x 200 ml of wash buffer C.
4. Wash 4x 200 ml of wash buffer D + BSA.
5. Resuspend beads in 200 ml of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of 1x buffer 4 + BSA, combine the content. Wash once more with 200 ml of 1x buffer 4 + BSA.
IV. Release of cDNA-tags using Tagging Enzyme MmeI
1. Prepare fresh 500 mM SAM by 1:64 dilution of 32 mM stock into dH2O.
2. Resuspend beads in the following mix and incubate at 37oC for 1 hour, mix every 10 min.
dH2O 150 ml
10x buffer 4 20 ml
500 mM SAM 20 ml
MmeI 10 ml
200 ml
3. Centrifuge at14 K for 2 min then transfer supernatant to a phase lock gel tube. Wash beads with 40 ml of LOTE, pull these together. Add 240 ml PC8, vortex and spin @ 14 K for 2 min.
4. Transfer aqueous layer to a new microcentrifuge tube. Take out 1/10 for no ligase negative control (if have multiple samples, ligase minus reactions can be pooled together) and add LoTE to 216 ml final volume. Ethanol precipitate:
Tags 216 ml
Glycogen 4 ml
3M NaOAc 22 ml
EtOH (ice cold) 650 ml
Centrifuge at 14 K, RT for 10 min.
5. Wash twice with ice cold 70% ethanol, spin again after last wash, and carefully remove residual liquid with pipette tip.
6. Resuspend pellet in 5 ml LoTE. The final volume of the pellet is ~5.5 ml.
V. Ligating Gex adapter 2
Gex Adapter 2:
5'-CAAGCAGAAGACGGCATACGANN-3’
3’-GTTCGTCTTCTGCCGTATGCT-phos-5’
1. add:
ligase plus mix ligase minus mix
3mM Tris pH 7.5 2 ml 3 ml
5x ligase buffer 2.4 ml 2.4 ml
Gex Adapter 2 (1.5 mM) 1 ml 1 ml
T4 ligase (high conc) 1 ml 0 ml
2. Incubate overnight at 16oC.
---------------------------------------------PAUSE POINTS---------------------------------------------------
VI. PCR amplification of tags
Gex PCR Primer 1:
5'-CAAGCAGAAGACGGCATACGA-3’
Gex PCR Primer 2:
5'–AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3’
1. Optimize PCR conditions (cycle number):
Ligation product 3 ml
5x Phusion HF buffer 10 ml
DMSO 1.5 ml
10 mM dNTP 1 ml
Gex PCR Primer 1+2 mix (1:5 – 5 mM) 1 ml
Phusion Hot Start DNA Polymerase 0.5 ml
dH2O 33 ml
50 ml
2. Perform PCR at following temperatures: 98oC, 2 min; 98oC, 15 sec; 60oC, 30 sec; 72oC, 15 sec (9 cycles). Remove 9 ml and perform PCR with the remaining reaction at following temperatures: 98oC, 15 sec; 60oC, 30 sec; 72oC, 15 sec (3 cycles). Repeat the above step 3x.
3. Run the product from each cycle number (9, 12, 15, 18, 21 cycles) on 12% polyacrylamide gel using 10 bp marker @ 250 V, 1 hr. Amplified tags should be ~85 bp. The cycle number that gives the optimal amplification varies with samples. Negative control (No ligase sample) should not contain any amplified product of the size of the tags.
VII. Gel purification of PCR product
1. After determining the optimal cycle number, repeat PCR (it is suggested to use the sub-peak cycle number and do 2x 50 ml reactions then pool together). Add 10 ml 5x FSB to 50 ml reaction and load into 1 well of a 10-well 12% polyacrylamide gel. Run 180 V for ~2.5 hr.
2. Cut out the 85 bp band and crush the gel (puncture the bottom of a 0.5-ml tube with a 18G needle, place it into a 2-ml tube, put gel into the 0.5 ml tube then cf @14K for 3 min). Elute fragment in 500 ml LoTE+acetate mix (9:1 of LoTE and 10 M NH4OAc) overnight @ 65oC.
3. Place the gel/LoTE mix (cut the pipette tip) into a spinX tube and cf @14K for 2x 5 min, switch the direction of tubes in between.
4. Extract with equal amount of PC8, transfer aqueous phase to a 1.5 ml tube and isopropanol precipitate:
Sample 500 ml
Glycogen 4 ml
2 M sodium perchlorate 167 ml
Isopropanol 500 ml
Vortex to mix, and cf @ 14K, 4oC, 30 min.
5. Wash 3x 1 ml 70% ethanol. Resuspend the pellet in 10 ml LoTE.
6. Measure with picogreen or nanodrop.
7. Submit for sequencing.
Gex Sequencing Primer:
5'-CCGACAGGTTCAGAGTTCTACAGTCCGACATG
I. cDNA synthesis
Note:
There are many mixing and washing steps of the magnetic beads throughout this protocol. Mix the beads by flicking or gentle vortex. Centrifuge briefly at low speed to collect beads stuck on the tube cap. Do washes by pipetting the beads up and down several times, esp. when clumping occurs. If beads stick to the sides of tubes, scrape with pipette tip to recover them. In all the washing steps, add solution to the tube while it's still on the magnetic stand in order to minimize drying of the beads, then close the cap, remove it from the magnet and resuspend beads. When removing supernatant, place the pipette tip at the opposite side of the tube, away from beads, slide the pipette tip to the bottom and pipette up very slowly, so you won't disturb and lose the beads.
1. Thoroughly resuspend the oligo(dT) beads, transfer 100 ml to a RNase-free 1.5-ml siliconized tube and place on magnet. After 30 sec remove supernatant.
2. Resuspend beads in 500 ml lysis/bindig buffer by gentle vortex. Leave beads in this buffer until ready to mix them with the cell lysate/RNA sample, remove it right before that.
3. If using cells as starting material, lyse cells in 1 ml of lysis/binding buffer, reduce viscosity by pressing it through a 21 G needle using a 1ml syringe. If using total RNA or mRNA, adjust the volume to 1 ml with lysis/binding buffer. Add lysate/RNA to pre-washed oligo(dT) beads and incubate at RT for 5 min with constant agitation (by hand).
4. Place the tube on magnet for 2 min, remove supernatant (the supernatant from cell lysate can be saved and used for DNA prep.).
5. Wash 2x 1 ml of wash buffer A, 1x 1 ml of wash buffer B, 4x 100 ml of 1x 1st strand buffer (dilute 5x 1st strand buffer with DEPC treated water).
6. Resuspend beads in 1st strand synthesis mix:
DEPC water 54.5 ml
5x 1st strand buffer 18 ml
0.1 M DTT 9 ml
10 mM dNTP 4.5 ml
RNaseOUT 1 ml
87 ml
7. Place the tube at 37oC for 2 min, then add 3 ml of SuperScript RT and mix. Incubate at 37oC for 1 hr, mix beads every 10 min by hand vortexing. After incubation place tube on ice to terminate the reaction.
8. On ice, add the components of the 2nd strand synthesis, in the order shown, to the first strand reaction:
1st strand reaction 90 ml
DEPC water (prechilled) 477 ml
5x 2nd strand buffer 150 ml
10 mM dNTP 15 ml
E. coli DNA ligase 3 ml
E. coli DNA polymerase 12 ml
E. coli Rnase H 3 ml
750 ml
Mix and incubate at 16oC for 2 hours, mix beads every 10 min.
9. After incubation place tubes on ice and terminate reaction by adding 45 ml of 0.5 M EDTA. Place the tube on magnet for 2 min, remove supernatant.
10. Preheat wash buffer C to 75C. Wash 1x 200 ml of wash buffer C, resuspend beads in 200 ml wash buffer C and heat at 75C for 20 min to inactive E. coli DNA polymerase.
11. Wash 4x 200 ml of wash buffer D + BSA. Take 2.5 ml of the last wash of wash buffer D to check the integrity of cDNA by RT-PCR [for 2x 25 ml reactions of β-actin & the other gene. Thermal cycle: 94C, 3 min; 94C, 15 sec, 64oC, 30 sec, 72C, 1 min/kb (3 cycels); 94C, 15 sec, 61C, 30 sec, 72C, 1 min/kb (3 cycels); 94oC, 15 sec, 58oC, 30 sec, 72C, 1 min/kb (35 cycels); 72oC, 5 min].
12. Resuspend beads in 200 ml of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of 1x buffer 4 + BSA, combine the content. Wash once more with 200 ml of 1x buffer 4 + BSA. The beads can be stored in 1x buffer 4 + BSA at 4oC O/N.
Note:
Use 2x BSA in all the washing buffers (20 ml 100x/ml). More BSA seems to reduce stickiness and improves the efficiency of the washes and the quality of the library. After the wash buffer C washing/heating step the beads are stickier until the first BSA wash, but then they are OK.
---------------------------------------------PAUSE POINTS---------------------------------------------------
II. Cleavage of cDNA with anchoring enzyme (NlaIII)
1. Resuspend beads in following mix and incubate at 37oC for 1 hour. Mix beads every 10 min.
dH2O 170 ml
100x BSA 4 ml
10x buffer 4 20 ml
NlaIII 6 ml
200 ml
2. Place the tube on magnet for 1 min, remove supernatant. Preheat wash buffer C to 37oC to prevent precipitation of SDS. Wash 2x 200 ml of wash buffer C to inactivate NlaIII.
3. Wash 1x 200 ml wash buffer D + BSA. Resuspend beads in 200 ml of wash buffer D + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of wash buffer D + BSA, combine the content. Wash 2x 200 ml wash buffer D + BSA. The beads can be very clumpy at this point, so be patient and keep pipetting them up and down until they are not clumpy.
4. Wash 2x 200 ml 1x ligase buffer. At final rinse transfer to a new siliconized tube.
III. Ligating Gex Adapter 1 to cDNA
Gex Adapter 1:
5'-ACAGGTTCAGAGTTCTACAGTCCGACATG-3’
3’-CAAGTCTCAAGATGTCAGGCT-phos-5’
1. Remove last wash and resuspend beads as follows:
dH2O 6 ml
5x ligase buffer 2 ml
Gex Adapter 1 (10 μM 1* ml
9 ml
*The amount of adapters used needs to be adjusted according to the number of cells.
2. Heat tubes at 50oC for 2 min. Let sit at RT for 10 min. Add 1 ml of T4 ligase (high conc.) to each tube and incubate at 16oC for 2 hours. Mix beads every 15 min.
3. Preheat wash buffer C to 37oC. Wash 2x 200 ml of wash buffer C.
4. Wash 4x 200 ml of wash buffer D + BSA.
5. Resuspend beads in 200 ml of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 ml of 1x buffer 4 + BSA, combine the content. Wash once more with 200 ml of 1x buffer 4 + BSA.
IV. Release of cDNA-tags using Tagging Enzyme MmeI
1. Prepare fresh 500 mM SAM by 1:64 dilution of 32 mM stock into dH2O.
2. Resuspend beads in the following mix and incubate at 37oC for 1 hour, mix every 10 min.
dH2O 150 ml
10x buffer 4 20 ml
500 mM SAM 20 ml
MmeI 10 ml
200 ml
3. Centrifuge at14 K for 2 min then transfer supernatant to a phase lock gel tube. Wash beads with 40 ml of LOTE, pull these together. Add 240 ml PC8, vortex and spin @ 14 K for 2 min.
4. Transfer aqueous layer to a new microcentrifuge tube. Take out 1/10 for no ligase negative control (if have multiple samples, ligase minus reactions can be pooled together) and add LoTE to 216 ml final volume. Ethanol precipitate:
Tags 216 ml
Glycogen 4 ml
3M NaOAc 22 ml
EtOH (ice cold) 650 ml
Centrifuge at 14 K, RT for 10 min.
5. Wash twice with ice cold 70% ethanol, spin again after last wash, and carefully remove residual liquid with pipette tip.
6. Resuspend pellet in 5 ml LoTE. The final volume of the pellet is ~5.5 ml.
V. Ligating Gex adapter 2
Gex Adapter 2:
5'-CAAGCAGAAGACGGCATACGANN-3’
3’-GTTCGTCTTCTGCCGTATGCT-phos-5’
1. add:
ligase plus mix ligase minus mix
3mM Tris pH 7.5 2 ml 3 ml
5x ligase buffer 2.4 ml 2.4 ml
Gex Adapter 2 (1.5 mM) 1 ml 1 ml
T4 ligase (high conc) 1 ml 0 ml
2. Incubate overnight at 16oC.
---------------------------------------------PAUSE POINTS---------------------------------------------------
VI. PCR amplification of tags
Gex PCR Primer 1:
5'-CAAGCAGAAGACGGCATACGA-3’
Gex PCR Primer 2:
5'–AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3’
1. Optimize PCR conditions (cycle number):
Ligation product 3 ml
5x Phusion HF buffer 10 ml
DMSO 1.5 ml
10 mM dNTP 1 ml
Gex PCR Primer 1+2 mix (1:5 – 5 mM) 1 ml
Phusion Hot Start DNA Polymerase 0.5 ml
dH2O 33 ml
50 ml
2. Perform PCR at following temperatures: 98oC, 2 min; 98oC, 15 sec; 60oC, 30 sec; 72oC, 15 sec (9 cycles). Remove 9 ml and perform PCR with the remaining reaction at following temperatures: 98oC, 15 sec; 60oC, 30 sec; 72oC, 15 sec (3 cycles). Repeat the above step 3x.
3. Run the product from each cycle number (9, 12, 15, 18, 21 cycles) on 12% polyacrylamide gel using 10 bp marker @ 250 V, 1 hr. Amplified tags should be ~85 bp. The cycle number that gives the optimal amplification varies with samples. Negative control (No ligase sample) should not contain any amplified product of the size of the tags.
VII. Gel purification of PCR product
1. After determining the optimal cycle number, repeat PCR (it is suggested to use the sub-peak cycle number and do 2x 50 ml reactions then pool together). Add 10 ml 5x FSB to 50 ml reaction and load into 1 well of a 10-well 12% polyacrylamide gel. Run 180 V for ~2.5 hr.
2. Cut out the 85 bp band and crush the gel (puncture the bottom of a 0.5-ml tube with a 18G needle, place it into a 2-ml tube, put gel into the 0.5 ml tube then cf @14K for 3 min). Elute fragment in 500 ml LoTE+acetate mix (9:1 of LoTE and 10 M NH4OAc) overnight @ 65oC.
3. Place the gel/LoTE mix (cut the pipette tip) into a spinX tube and cf @14K for 2x 5 min, switch the direction of tubes in between.
4. Extract with equal amount of PC8, transfer aqueous phase to a 1.5 ml tube and isopropanol precipitate:
Sample 500 ml
Glycogen 4 ml
2 M sodium perchlorate 167 ml
Isopropanol 500 ml
Vortex to mix, and cf @ 14K, 4oC, 30 min.
5. Wash 3x 1 ml 70% ethanol. Resuspend the pellet in 10 ml LoTE.
6. Measure with picogreen or nanodrop.
7. Submit for sequencing.
Gex Sequencing Primer:
5'-CCGACAGGTTCAGAGTTCTACAGTCCGACATG