GSM2564046: Tau 17-DMAG 1nM rep1 b3; Homo sapiens; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Neural
Cell type
Forebrain neurons
NA
NA
Attributes by original data submitter
Sample
source_name
forebrain neurons_Tau 17-DMAG 1nM
tissue/cell type
Forebrain neurons
overexpression
MAPT
treatment
17-DMAG 1nM + DMSO
batch
Batch 3
number of read pairs
6178867
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
ATAC-seq was performed as previously described by Buenrostro et al. (PMID: 24097267) with three modifications: (i) To reduce mitochondrial DNA contamination, we substituted 0.1% Ipegal with 0.01% Tween in the nuclear lysis buffer. (ii) Additional tagmentation buffer (TD; 2x) was prepared as follows: 20 mM Tris(hydroxymethyl)aminomethane and 10 mM MgCl2; adjusted to pH 7.6 with 100% acetic acid before addition of 20% (vol/vol) dimethylformamide (Wang et al., 2013). (iii) We performed the PCR amplification in a 25ul total volume rather than 50ul to save half of our tagmented DNA for backup. After 31 days in vitro, iPSC neurons were trypsinised and counted to provide 50,000 live cells for ATAC-seq. iPSC-derived neurons were lysed with 0.01% Tween, 10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and nuclei pelleted, washed and transposed upon addition of 2.5 uL Nextera Tn5 Transposase in 1x TD buffer (Nextera Kit, Illumina) and incubation at 37°C for 30 mins. Immediately following transposition, DNA fragments were purified using a Qiagen MinElute PCR Purification Kit (Qiagen) following the manufacturer’s instructions. Transposed DNA was amplified with dual indexed Nextera PCR primers for 5 cycles prior to removing a 5ul aliquot from each sample to test further amplification requirements by qPCR. Rounding up the cycle threshold from each sample decides how many additional cycles each sample needs to be amplified. For these samples, we needed to re-array and continue amplifying the remaining 20ul for 5-8 cycles. Final libraries were cleaned twice with 1.5X Ampure XP SPRI beads and eluted in 12ul of water.