Reagents
Plasmids
NTT-seq plasmids are available from AddGene: https://www.addgene.org/browse/article/28225051
Custom sequencing primers
Custom sequencing primers required for NTT-seq can be ordered from IDT or any DNA sythesis company. The following primer sequences are required, and can be ordered as standard oligos:
Custom read 1 primer (5'-3'):
GCGATCGAGGACGGCAGATGTGTATAAGAGACAG
Custom index 2 primer (5'-3'):
CTGTCTCTTATACACATCTGCCGTCCTCGATCGC
Barcoded Tn5 adapters
Custom Tn5 adaptors are used in the NTT-seq protocol that incorporate a short DNA barcode. The base structure of these custom adapters are:
5'- TCGTCGGCAGCGTC NNNNNNNN GCGATCGAGGACGGCAGATGTGTATAAGAGACAG -3'
Where NNNNNNNN represents the 8-bp Tn5 barcode. These adaptors can be ordered from
IDT or any DNA synthesis company as standard DNA oligos. Before loading Tn5 with adaptors,
the barcoded top-strand MEDSA adaptor (above) will need to be annealed to the lower-strand
adaptor, which has the following sequence:
5'- /Phos/CTGTCTCTTATACACATCT -3'
Note that this oligo requires a 5' phosphate group.
The barcoded MEDSA oligo should be combined 1:1 with standard (not barcoded) MEDSB, which has the following sequence:
5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
Starter kits
NTT-seq starter kits with enough reagents for 8 NTT-seq experiments can be requested from the Center for Integrated Cellular Analysis website: https://www.multimodalintegration.org/techsharing/
The starter kit contains:
- Purified rabbit and mouse nb-Tn5 enzyme loaded with barcoded adaptors
- Custom sequencing primers for 10 sequencing runs
- H3K27me3 (mouse) and H3K27ac (rabbit) primary antibodies
If you received an NTT-seq kit, the following Tn5 barcodes were used:
- Anti-mouse nb-Tn5:
GTCAAGGA - Anti-rabbit nb-Tn5:
CAGAGCGT