Hi everyone,

I'm trying to perform ATAC-seq without using any commercial kit, but it's been challenging to find a protocol that aligns with this approach. I’d like to ask if anyone has experience with this.

Here’s the workflow I followed:

1. Transposon Annealing:

ME A: 5'-(index sequence)AGATGTGTATAAGAGACAG-3' ME B: 5'-(other index sequence)AGATGTGTATAAGAGACAG-3' ME rev: 5'-pCTGTCTCTTATACACATCT-3' By following the standard primer annealing protocol, I obtained two transposon oligos (ME A-rev, ME B-rev).

2. Nuclei Extraction: I followed the Kaestner Lab Omni ATAC protocol (file attached) and confirmed the extraction by performing MNase digestion. This step seems to be working fine.

3. Transposition:

I've attached the protocol for the Tn5 transposase I used. Following this protocol and the Omni ATAC protocol, I set up the transposition reaction with the following conditions:1 µl 10X Tn5 Transposase Buffer (final conc. 1X) 1 µl Annealed Transposon A (final conc. 1 µM) 1 µl Annealed Transposon B (final conc. 1 µM) 0.1 µl 10% Tween-20 0.1 µl 1% Digitonin 1 µl Tn5 transposase Add D.W. to 10 µl I added this reaction mix to the extracted nuclei and incubated at 37°C for 2 hours.

4. DNA Purification: I purified the DNA using the QIAquick PCR purification kit.

After these steps, I performed PCR for amplification and confirmed the product with gel electrophoresis, looking for bands at 300, 450, and 600 bp (corresponding to mono-, di-, and trinucleosomes, with ~150 bp added for index and adapter sequences).

This method worked once, but I haven't been able to replicate the results since then. Instead of the expected bands, I could only observe smears or odd bands after PCR (figures attached). I would really appreciate any advice or insights you might have.

Sincerely, Hyelin