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Old 03-13-2019, 01:12 AM   #8
pritb
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Location: Singapore

Join Date: Mar 2019
Posts: 2
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Hello All

I am new to ATAC seq experiments. I am working on human fibroblast with 50k cells (counted through countess). Initially, I started following J Buenrostro (2015) protocol but seems not working. Now I shifted myself to Omni-ATAC protocol.

My Initial OD (after Tn5 treatment) using Nanodrop gave 46 ng/ul. I proceeded with Library prep by giving 5 cycles. Realtime PCR for library quantification gave me 20 additional cycles for my Libraries. After PCR cleanup, I got 96 ng/ul as library concentration. On gel check, I cant seen anything, except for few bands that is less than 100 bps. What could be reason?

I have attached my gel picture here.
Lane-1: 2 ul of sample after Tn5 reaction post cleanup
Lane-2: 15 ul of sample after 35 cycles (Although I selected 20 cycles based on 1/4 fluorescence intensity)
Lane-3: 10 ul of sample of 20 cycles (additional PCR cycles). This is purified. You can seen 2 bands less than 100 bps.
Attached Images
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