Brandon:LabNotes/Project1/2013-12-3: Difference between revisions
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'''SAMPLES (ALL), 55c 10 MINUTES''' | '''SAMPLES (ALL), 55c 10 MINUTES''' | ||
*add the below into one tube and incubate for 10 minutes at 55C. | *add the below into one tube and incubate for 10 minutes at 55C. | ||
.5 uL nextera LMW buffer | |||
2 uL lysed/pure genomic DNA (X ng/pg DNA) | 2 uL lysed/pure genomic DNA (X ng/pg DNA) | ||
0 uL Nuclease free water | |||
1 uL prepared and diluted T7 transposomes (MAKE SURE TO ADD LAST) | |||
'''OR DILUTED TSPN, 4X, 8X, 16X''' | '''OR DILUTED TSPN, 4X, 8X, 16X''' | ||
___________ | ___________ | ||
5 uL total solution | 3.5 uL total solution | ||
5. Protease digestion of transposase, protease inactivation | 5. Protease digestion of transposase, protease inactivation | ||
To each tube, add: | To each tube, add: | ||
.5 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 | Incubate: 50C 10 minutes, 70C 20 minutes | ||
6. T4 ligation of T7 promoter and barcodes. | 6. T4 ligation of T7 promoter and barcodes. | ||
add sc-T7-R1-IdxXX to each sample | .5 uL add sc-T7-R1-IdxXX to each sample | ||
.5 uL of T4 DNA ligase. | |||
.5 uL T4 buffer (5X?) | |||
Incubate at 20C for 10 minutes | |||
7. Fill in reaction | |||
*Add | *Add 5 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera) | ||
8. Pool samples and clean with standard EtOH precipitation | |||
9. Maxiscript (Ambion) T7 Protocol, IVT | |||
*DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification. | *DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification. | ||
Revision as of 22:51, 4 December 2013
Transposon for single cell accessibility
- Know that transposon works well for 1000 cells and produces alot of RNA product after IVT amplification. However there are limitations to amplification by IVT, as ~400 pg of DNA is needed for amplification by IVT. To try and overcome this limitation, single cells can be barcoded upon tranposition, then pooled for IVT amplification.
- no cleaning steps required
- dilute transposome, found can still work well with 8X dilution. (still waiting for sequencing results on other dilution. However this is for 500 cells.
- There are two papers examining single cell transcriptomics with relevant methods.
- Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity
- use IVT for labeling, but PCR for amplification of cDNA. Single cells are isolated with FACS into a plate with .4 uL of lysis buffer that has a bubble, to ensure the cell is captured in the solution.
- CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification
- pools samples are performing reverse transcription and second strand synthesis to create a DNA template for T7. ~400 pg of total sample is needed for a viable T7 amplification. Will pool enough cells to obtain this amount of template.
- Tested IVT amplification on 50 cells and found enough RNA (~50 ng is generated to perform the barcoding reaction). however since barcoding is already done, downstream processing will be different.
- Thus 50-100 cells pooled after barcoding looks feasible.
- Possible issues
- loss of product when pooling samples. more efficient way to pool?
- Need to use DNA lo-bind tubes
- T4 ligation efficiency?
Single cell transposon design
sc-ME-top 5'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-bottom 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5' sc-T7-R1-IdxXX 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC -3' Complete sequence of transposome after ligation T7 consensus sequence (27) N2 Index seq (26) bc(6) for ligation/R1 (20) ME end (19) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' After annealing sc-ME-annealed 5'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5' sc-ME-bottom After transposition (fragment from two adjacent insertions) 5'- |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3' 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG -5' Single insertion 5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' 3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' After ligation with T4 (amp ligase?) (single insertion) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' sc-ME-top + sc-T7-R1-Idx49 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' sc-ME-bottom After gap fill in/extension with taq2x 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- TTTAATTATGCTGAGTGATATCCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' After IVT, RNA generated RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer. Extension with BST or klenow exo- or DNAP1 or Taq2x DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' (sc-SSS-prmr) cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
Normal Nextera 5' end: 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' ("A-METS" NXTA_ME_A) 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") after fill in: 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' PCR amplification: 5'-AATGATACGGCGACCACCGA-3' (NXTA_P1) 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' (NXTA_adpt1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
Custom Nextera transposome for 3' end 5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS") 3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' (sc-ILA-adaptor) 3' ILA adaptor 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") 3'- [CAAGCAGAAGACGGCATACGAGAT]CGAGATGTGTATAAGAGACAG -3' (sc-ILA-adaptor) 3' ILA adaptor After custom nextera tagmentation: Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' After fill in: DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCGATCTCGTATGCCGTCTTCTGCTTG -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' Amplification: ILA 5' adaptor 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGA sc-T7-PCR1 (comp part (NEB Tm=72), all (NEB Tm=83C) 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCA sc-T7-PCR2 (comp part (Tm=68), all (Tm=81C)) ILA 3' adaptor DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' 3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2) (NEB Tm=65C) 3'- GC[TAGAGCATACGGCAGAAGACGAAC -5 (NXTA_P2_E) (NEB Tm=70C) Read primers (N2 Index read) T7tspn-Read1 (Tm=75.80) ILA 5' adaptor AAGAGCGGTTCAGCAGGAATGCCGAG TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG ILA 3' adaptor 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCT]CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'
Protocol
IVT Protocol
- If need to make more transposome, do first 2 steps. If not goto step 3.
Generation of sc transposome
1. annealing of ME sequence top and bottom
- a. Make 100 uM stock solution of sc-ME-top and sc-ME-bottom.
- b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
- c. cool to RT at 0.1 C/s
2. transposome complex generation, run controls!!!
- add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
Generation of 3' ILA adaptor transposome
1. annealing of ME sequence to Nextera transposon sequence
- a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (sc-ILA-adaptor).
- b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
- c. cool to RT at 0.1 C/s
2. Transposome complex generation, run controls!!!
- add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed Nextera transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
3. Prepare samples, lyse cells with lysis buffer
sample list 1. XX 2. XX
CELL SAMPLES (ALL)
- 1. refrigerate and warm LB.
- 2. add warm or cold lysis buffer to cell aliquots, mix briefly
- 3. transposition ready
- Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
- spin down cells to concentrate them as necessary.
- Prepare 2X LB from 10X buffer. mineral oil optional.
- if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer
4. transposition reaction, using (sc transposon)
SAMPLES (ALL), 55c 10 MINUTES
- add the below into one tube and incubate for 10 minutes at 55C.
.5 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 0 uL Nuclease free water 1 uL prepared and diluted T7 transposomes (MAKE SURE TO ADD LAST) OR DILUTED TSPN, 4X, 8X, 16X ___________ 3.5 uL total solution
5. Protease digestion of transposase, protease inactivation
To each tube, add: .5 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
6. T4 ligation of T7 promoter and barcodes.
.5 uL add sc-T7-R1-IdxXX to each sample .5 uL of T4 DNA ligase. .5 uL T4 buffer (5X?) Incubate at 20C for 10 minutes
7. Fill in reaction
- Add 5 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
8. Pool samples and clean with standard EtOH precipitation
9. Maxiscript (Ambion) T7 Protocol, IVT
- DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
CLEAN WITH ZYMO RNA CLEAN AND CONCENTRATOR NOW
8. Clean RNA with AMPURE RNA beads. elute in 20 uL.
- Protocol changes:
- 1. add 30 uL to IVT reaction to bring to 50 uL total before starting protocol.
- 2. Use 1.8:1 ratio of beads, thus 80 uL of beads for 50 uL of sample in this case
- 3. Drying time after the washes is 3 minutes.
- elute in 20 uL and concentrate to 10 uL
- can quantitate with Qubit or on TBU gel.