Need help in ATAC-seq
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.
Can anyone help me out?
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.
Can anyone help me out?