Min Hu/Polyak Lab

MicroSAGE

This protocol is optimized for 5x104-2x106 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.

Prepare Oligo(dT) beads, lysis/binding buffer, prechilled DEPC treated H2O, RNAase free tubes, wash buffer A/B/C/D, 1X buffer 4, 1x1st strand buffer (from 5x 1st strand buffer with DEPC H2O), 100 x BSA, 16oC and 75oC heat block

  1. Thoroughly resuspend the oligo(dT) beads, transfer 100 l to a RNase-free 1.5-ml siliconized tube and place on magnet. After 30 sec remove supernatant.
  1. Resuspend beads in 500 l lysis/bindig bufferby gentle vortex. Leave beads in this buffer until ready to mix them with the cell lysate/RNA sample, remove it right before that.
  1. 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).
  1. Place the tube on magnet for 2 min, remove supernatant (the supernatant from cell lysate can be saved and used for DNA prep.).
  1. Wash 2x 1 ml of wash buffer A, 1x 1 ml of wash buffer B, 4x 100 l of 1x 1st strand buffer (dilute 5x 1st strand buffer with DEPC treated water).
  1. Resuspend beads in 1st strand synthesis mix:

DEPC water54.5 l

5x1st strand buffer 18 l

0.1 M DTT 9 l

10 mM dNTP 4.5 l

RNaseOUT 1 l

87 l

  1. Place the tube at 37oC for 2 min, then add 3 l 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.
  1. On ice, add the components of the 2nd strand synthesis, in the order shown, to the first strand reaction:

1st strand reaction 90 l

DEPC water (prechilled)477 l

5x2nd strand buffer150 l

10 mM dNTP 15 l

E. coli DNA ligase 3 l

E. coli DNA polymerase 12 l

E. coli Rnase H 3 l

750 l

Mix and incubate at 16oC for 2 hours, mix beads every 10 min.

  1. After incubation place tubes on ice and terminate reaction by adding 45 l of 0.5 M EDTA. Place the tube on magnet for 2 min, remove supernatant.
  1. Preheat wash buffer C to 75oC. Wash 1x 200 l of wash buffer C, resuspend beads in 200 l wash buffer C and heat at 75oC for 20 min to inactive E. coli DNA polymerase.
  1. Wash 4x 200 l of wash buffer D + BSA. Take 2.5 l of the last wash of wash buffer D to check the integrity of cDNA by RT-PCR [for 2x 25 l reactions of β-actin & the other gene. Thermal cycle: 94oC, 3 min; 94oC, 15 sec, 64oC, 30 sec, 72oC, 1 min/kb (3 cycels); 94oC, 15 sec, 61oC, 30 sec, 72oC, 1 min/kb (3 cycels); 94oC, 15 sec, 58oC, 30 sec, 72oC, 1 min/kb (35 cycels); 72oC, 5 min].
  1. Resuspend beads in 200 l of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 l of 1x buffer 4 + BSA, combine the content. Wash once more with 200 l 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 l 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------

Day 2

Preparation: Wash buffer C (37oC), wash buffer D, ligase buffer, 1x buffer 4, 37oC, 50oC, 16oC

II. Cleavage of cDNA with anchoring enzyme (NlaIII)

  1. Remove buffer 4+BSA. Resuspend beads in following mix and incubate at 37oC for 1 hour. Mix beads every 10 min.

LOTE170 l

100x BSA 4 l

10x buffer 4 20 l

NlaIII 6l

200 l

  1. Place the tube on magnet for 1 min, remove supernatant. Preheat wash buffer C to 37oC to prevent precipitation of SDS. Wash 2x 200 l of wash buffer Cto inactivate NlaIII.
  1. Wash 1x 200 l wash buffer D + BSA. Resuspend beads in 200 l of wash buffer D + BSA, transfer to a new siliconized tube. Wash the old tube with 200 l of wash buffer D + BSA, combine the content. Wash 2x 200 l 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.
  1. Wash 2x 200 l 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:

LOTE6 l

5xligase buffer2 l

Gex Adapter 1 (10 μM)1* l

9 l

*The amount of adapters used needs to be adjusted according to the number of cells.

  1. Heat tubes at 50oC for 2 min. Let sit at RT for 10 min. Add 1 l of T4 ligase (high conc.) to each tube and incubate at 16oC for 2 hours. Mix beads every 15 min.
  1. Preheat wash buffer C to 37oC. Wash 2x 200 l of wash buffer C.
  1. Wash 4x 200 l of wash buffer D + BSA.
  1. Resuspend beads in 200 l of 1x buffer 4 + BSA, transfer to a new siliconized tube. Wash the old tube with 200 l of 1x buffer 4 + BSA, combine the content. Wash once more with 200 l of 1x buffer 4 + BSA.

IV. Release of cDNA-tags using Tagging Enzyme MmeI

  1. Prepare fresh 500 M SAM by 1:64 dilution of 32 mM stock intodH2O (2 ul in 126 ul H2O).
  1. Resuspend beads in the following mix and incubate at 37oC for 1 hour, mix every 10 min.

LOTE150 l

10x buffer 4 20 l

500 M SAM 20 l

MmeI 10 l

200 l

  1. Centrifuge at 14 K for 2 min then transfer supernatant to a phase lock gel tube. Wash beads with 40 l of LOTE, pull these together. Add 240 l PC8, vortex and spin @ 14 K for 2 min.
  1. 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 l final volume. Ethanol precipitate:

Tags 216 l

Glycogen 4 l

7.5M NH4OAc 144 l

EtOH (ice cold) 1080l

Centrifuge at 14 K, RT for 10 min.

Wash twice with 500 lice cold 70% ethanol, spin again after last wash, and carefully remove residual liquid with pipette tip.

  1. Resuspend pellet in 6 l LoTE.

V. Ligating Gex adapter 2

Gex Adapter 2:

5'-CAAGCAGAAGACGGCATACGANN-3’

3’-GTTCGTCTTCTGCCGTATGCT-phos-5’

  1. add:

ligase plus mixligase minus mix

DNA 6 l 6 l

5x ligase buffer2 l2 l

Gex Adapter 2 (1.5 M)1 l1 l

T4 ligase (high conc) 1 l0 l

Total 10 l

  1. 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 product3 l

5x Phusion HF buffer10 l

DMSO1.5 l

10 mM dNTP7.5l

Gex PCR Primer 11 l

Gex PCR Primer 21 l

Phusion Hot Start DNA Polymerase1 l

dH2O 25l

50 l

If use platinum taq, use 10x platinum Taq buffer 5 l, 3l DMSO and 0.5l Platinum Taq and 32 l H2O.

2.Perform PCR (use the SAC1 program) at following temperatures: 94oC, 1 min; 94oC, 20 sec; 58oC, 30 sec; 72oC, 15 sec (9 cycles). Remove 9 l 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.

  1. 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 l reactions then pool together). Add 10 l 5x FSB to 50 l reaction and load into 2 well of a 10-well 12% polyacrylamide gel. Run 241 V for 50 min.

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 l 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 (Costar) and cf @14K for 2x 5 min, switch the direction of tubes in between.

  1. Extract with equal amount of PC8, transfer aqueous phase to a 1.5 ml tube and isopropanol precipitate:

500 l sample

4 l glycogen

167 l 2 M sodium perchlorate

500 l isopropanol

Vortex to mix, and cf @ 14K, 4oC, 30 min.

  1. Wash 3x 1 ml 70% ethanol. Resuspend the pellet in 10 l LoTE.
  2. Measure with picogreen or nanodrop.
  3. Submit for sequencing.

Gex Sequencing Primer:

5'-CCGACAGGTTCAGAGTTCTACAGTCCGACATG

SAGE buffers

Stock 1M Tris-HCl

5M NaCl

10% SDS

0.1M DTT

0.5M EDTA

20 mg/ml Glycogen

1.5M LiCl (636 mg in 10 ml dH2O)

5M Tris (6.06g/10 ml in dH2O)

Wash Buffer A (For 50 ml)

500 ul Tris-HCl

5 ml LiCl

100 ul EDTA

50 mg Lithium dodecyl sulfate

25 ul glycogen

Add dH2O to make up

Wash Buffer B (50 ml)

500 ul Tris-Hcl

5 ml LiCl

100 ul EDTA

25 ul glycogen

add dH2O to make up to 50 ml

Wash Buffer C (50 ml)

50 ul Tris

50 ul EDTA

10 ml NaCl

5 ml 10% SDS

25ul glycogen

Wash Buffer D (50 ml)

50 ul Tris

50ul EDTA

10 ml NaCl

10 mg BSA

Lysis/Binding Buffer (50 ml)

5 ml Tris-HCl (PH 7, 1N)

5ml 5M LiCl

1 ml 0.5m EDTA (PH 8)

25 ml 2% Lithium dodecyl sulfate (LDS)

2.5 ml 0.1M DTT

11.5 ml DEPC treated H2O