Brandon:LabNotes/Project1/2013-12-3
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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 GCGCCATCAG][AGATGTGTATAAGAGACAG] sc-ME-bottom TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] sc-T7-R1-Idx49 [AAATTAATACGACTCACTATAGGGAGA][TTCTCGCCAAGTCGTCCTTACGGCTC][BARCOD][ATCCTCCCTC Complete sequence of transposome after ligation T7 consensus sequence (27) N2 Index seq (26) (6) for ligation/R1 (20) ME end (19) [AAATTAATACGACTCACTATAGGGAGA][TTCTCGCCAAGTCGTCCTTACGGCTC][BARCOD][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] After annealing sc-ME-annealed GCGCCATCAG][AGATGTGTATAAGAGACAG] sc-ME-top TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] sc-ME-bottom After ligation with T4 (amp ligase?)