Brandon:LabNotes/Project1/2012-6-6

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RNA fragmentation protocols, RNase III, ([see Mg++ protocol here])


  • After IVT with T7, RNA generated and needs to be fragmented.
  • protocol for IVT RNA -> RNase III fragmentation -> PolyA polymerase (MMLV buffer) -> MMLV RT -> PCR amplification


  • possible issues
    • run with and without 5' blocking primer for DNA-RNA hybrid.
    • might need to purify after T7 IVT, since [Tris Hcl] is so much higher.
1X T7 buffer:
400 mM Tris Hcl
8  mM MgCl2
2  mM spermidine-Hcl
25 mM NaCl
PH 7.9

1X NEBNext RNase III Reaction Buffer: 
10 mM Tris-HCl 
10 mM Mg(Cl)2 
1 mM DTT 
60 mM NaCl 
pH 8.3 @ 25°C

10X Poly(A) Polymerase buffer:
500 mM Tris-HCl
2.5 M NaCl
100 mM MgCl2
pH 7.9 @ 25°C

MMLV
Invitrogen (though using clontech)
5X First-Strand Buffer
250 mM Tris-HCl (pH 8.3 at room temperature
375 mM KCl
15 mM MgCl2
0.1 M DTT


Rnase III protocol

1. Retrieve IVT RNA.

  • samples:
1. with Blocking Primer, protease treated
2. with blocking primer, EtOH cleaned
3. NTC with blocking primer, protease
4. without blocking primer, protease treated
5. without blocking primer, EtOH cleaned
6. NTC without blocking primer, protease treated

used 5 uL RNA for each sample


2. Perform EtOH cleanup of RNA. (can do columns)


3. Perform RNase III fragmentation (NEB):

Starting Material: Purified mRNA (50–250 nanograms)

 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid.
 *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2.
 *b. Incubate at 95C for 2 minutes.
 *c. cool to RT at 0.1 C/s


2. Mix the following components in a sterile PCR tube:

 X  uL Purified mRNA + blocking primer (50-250 nanograms)
 .5  uL RNase III (1 unit/μl)
 1   uL RNase III Reaction Buffer (10X)
 5.5 uL Nuclease-Free Water
 add in DNA primer to protect 5' end since don't want degradation??
 _______
 10 uL total volume


3. Incubate in a preheated thermal cycler for 5 minutes at 37°C.

4. Transfer tube to ice.


4. (EtOH clean up OR RNA column purification) OR protease digestion

  • EtOH cleanup
a. Add 80 μl of cold Nuclease-Free water. (brings to 100 ul)

b. Add 3 volumes 100% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained.
  In this case:
  10   uL NaOAc (mix after adding)
  300  uL  100% EtOH
  1    uL Glycol Blue

c. Chill solution in -80 for 30 minutes, cool centrifuge to 4C

d. Spin at 4C for 20 minutes at max speed. Chill 75% EtOH. 

e. Blue pellet should be visible, Discard supernatent.

f. Wash the pellet with cold 75% EtOH

h. Air dry pellet for up to 10 minutes at room temp to remove residual EtOH

j. Resuspend in appropriate volume in nuclease free H2O.
  • Protease digestion
To each tube, add:
1 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


  • Run TBU gel to see fragmentation profile


5. Poly(A) Addition with polyA polymerase (Enzymatics)

  • enzymatics PolyA polymerase.
1. assemble reaction:
   2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer)
   1 uL polyA enzyme
   1 uL 10 mM ATP
   bring to 10 uL with RNA or w/e

2. Incubate at 37C for 10 minutes

3. Heat inactivate at 70C for 20 minutes. (rui and NEB)



6. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O (one from BENG160 class, T20VN_PE_R)

2. heat the mixture to 70C fo 3 minutes.  Immediately cool on ice.

3. Add the following to the reaction.
   2  uL 5X first strand buffer
   3  uL dNTP mix
   3  uL 100 uM DTT
  .75 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ____
   32.5 uL total

4. Incuvate at 42C for 60 minutes

5. Terminate the reaction by heating at 70C for 10 minutes



7. second strand synthesis (qPCR) (KAPA)

KAPA SYBR FAST qPCR mix X35 cycles

25 uL KAPA SYBR
4    uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24))
1  uL H2O
20    uL DNA template (use whole RT reaction)

KAPA SYBR cycles:
98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever

  • terminate when curves saturate


8.qiaquick cleanup

  • run on gel or w/e
  • run qiaquick to clean sample before performing qPCR.
    • 6 enzymes from 6 different reactions in there already.

Results

  • TBU gel after RNase III fragmentation and purification with EtOH or enzyme inactivation with protease
  • smears still present

File:ZhangLab 2 2012-06-07 18hr 02min-labled.jpg


  • result from PCR amplification
  • odd how samples 1,2 and 3,4 have almost the same smear even though only sample 1 had high amplification readings during PCR.

File:2012-06-12 qPCR after rnase III with plate.bmp


  • TBE gel after RT, PCR amplification
  • smears look good, can size select and proceed to cloning/transformation to check for correct inserts.

File:ZhangLab 2 2012-06-12 16hr 35min-labeled.jpg


  • TBU gel after RT, PCR amplification

File:ZhangLab 2 2012-06-12 18hr 02min-labeled.jpg


  • gel size selection of band, 250 bp to 1000 bp.

File:ZhangLab 2 2012-06-13 15hr 14min-labeled.jpg


  • gel after size selection. used 4 uL total of PCR amplified product, resuspended in 10 uL of nuclease free H2O. Have about 50% sample loss from gel size selection. Original samples have a total of 122.28 ng/4 uL, and 102.68 ng/4 uL. Except sample 1, lost alot of sample somehow. probably pipetting or something.
  • quanitation from size selection gel.
smpl1 0.72 ng/ul in 10 uL
smpl2 4.78 ng/ul in 10 uL
smpl4 6.08 ng/ul in 10 uL
smpl5 4.8 ng/ul in 10 uL
7.2 ng/total
47.8 ng/total
60.8 ng/total
48 ng/total

File:ZhangLab 2 2012-06-13 19hr 06min-labeled.jpg



  • Continuing with cloning and transformation etc. to check if has good genomic DNA inserts
  • For ligation, Used 3 uL DNA for sample 1. Used 1 UL of DNA for the other samples since they have 5 ng/ul already
  • send in sample 1,7,18,20,21,22,23 for sequencing, could be insert, just very low efficiency
  • PCRed 11 white colonies for each sample
  • cloning insert information:
see information for sequences of top2 for sequences
85  bp = size of end with polyA, adaptor 2 (3' end)
46+15 bp = size of 5' end of transposon (+15 since ME sequence is still after primer) (5' end)
_______
146

M13 Priming sites
89  bp until insertion from M13 reverse (top strand, is fwd in sequencing)
111 bp until insertion from M13 Forward (bottom strand, is reverse in sequencing)
____
200 bp

Thus if 200+146 = 346 bp or larger fragment after PCR amplification, then it has an insert.  For size selected
insert, needs to be 346+250 = 596 bp to contain a correctly size selected insert.


M13 Reverse sequence (beginning)
[CAGGAAACAGCTATGAC]CATGATTACGCCAAGCTTGGTACCGAGCTCGTC[GGATCCACTAGTAAC]GGCCGCCAGTGTGCTGGAATTCGGCTT
 M13 reverse primer                                  usually starts here for sequencing


M13 Forward sequence (end) (plasmid sequence)
GCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCC
TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTT
ACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCG
CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG
CTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT
TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTA
GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC
TCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC
CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATTC
ANGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCG
GATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGAT


  • 1. with Blocking Primer, protease treated

File:ZhangLab 2 2012-06-15 16hr 13min-labeled.jpg

  • 2. with blocking primer, EtOH cleaned

File:ZhangLab 2 2012-06-15 16hr 15min-labeled.jpg

  • 4. without blocking primer, protease treated

File:ZhangLab 2 2012-06-15 16hr 36min-labeled.jpg

  • 5. without blocking primer, EtOH cleaned

File:ZhangLab 2 2012-06-15 16hr 38min-labeled.jpg


  • 2nd set
  • for ligation, used 4x the amount of DNA. used 4 UL per tube, opposed to the 1 uL per tube in first transformation. will see if results differ.
  • send in samples 2,4,6,9,14,17 for sequencing. few looks like could have a good insert.
  • PCR'ed 8 colonies for each sample


  • 1. with Blocking Primer, protease treated

File:ZhangLab 2 2012-06-17 17hr 58min-labeled.jpg

  • 2. with blocking primer, EtOH cleaned

File:ZhangLab 2 2012-06-17 17hr 59min-lableled.jpg

  • 4. without blocking primer, protease treated

File:ZhangLab 2 2012-06-17 18hr 11min-labeled.jpg

  • 5. without blocking primer, EtOH cleaned

File:ZhangLab 2 2012-06-17 18hr 12min-labeled.jpg



  • CLoning stats:
first round ' BP No BP
protease 3 1
EtOH 3 0
second round BP No BP
protease 2 0
EtOH 3 1
11 2
  • Blocking primer is definetly helping, since that is the only difference between the two.


  • 4 have verified inserts, matches 100% to human genome
    • except 1 which has some SNPs since insert is flipped and polyA sequence is sequenced first in the sequencing run, thus disrupting reads after.
  • 5 are disrupted since polyT is in reverse orientation so harder to sequence, but prolly have viable inserts based on size from PCR gel.


  • Not sure why the cloning isn't working as well. Could be bad previous amplifications or TA tailing. Can take PCR amplified DNA, add taq polymerase 2X, at 72C for 30 minutes to add A tails for sure, then beads purification, and cloning/transformation again.

Notes and primers used etc

Blocking primer for protecting 5' end from RNase III degradation

T7-frag-block
5'- CTGTCTCTTATACACATCTTAATGTCTCCC/3InvdT/

3InvdT so don't get any nonspecific extension by MMLV RT.

Stuff used for RNA-seq for BENG160 class

5’ end addition primers

TSO_N10_BC[XX]
[AAGCAGTGGTATCAACGCAGAGdUdU]NNNNNNNNNNTTTAGGrGrGrG
         adatpor1

5'- AAG CAG TGG TAT CAA CGC AGA G/ideoxyU//ideoxyU/ NNN NNN NNN NTT TAG GrGrGrG -3'

P1-STRT
5’- [AATGATACGGCGACCACCGA][GATCT][AAGCAGTGGTATCAACGCAGAGT] -3’  (Tm=82.59)
      ILA adaptor blue Tm=64.31         adaptor1 Tm=61.02

STRT-SEQ
5'- [GATCT][AAGCAGTGGTATCAACGCAGAGTT] -3'
                adaptor1
3’ end addition primers

T20VN_PE_R
5'-Bio-[GCATTCCTGCTGAACCGCTCTT]CCGATCTTTTTTTTTTTTTTTTTTTTTVN  -3’  (Tm=78.92)
          adaptor2 Tm=65.68

PCR_R.N2Ind[XX] (XX=23,24 for me)
5’- [CAAGCAGAAGACGGCATACGAGAT][TACAAG]CTCG][GCATTCCTGCTGAACCGCTCTT] -3’ (Tm=87.12)
        ILA adaptor orange       bc          adaptor2 Tm=65.68
My modifications for T7-tspns for 5' addition primers
 
To use with (T7-top and T7-top2) transposons

5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT]  -3’  (Tm=86.59)
     ILA adaptor blue           Top2-5’end (T7-top2-PCR-iaf) Tm=66.79


To use with (T7-top3) transposon

5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’  (Tm=81.40)
     ILA adaptor blue              Top3-5’end (T7-top3-PCR-iaf) Tm=60.71