alias: GSM3831242
title: GSM3831242: Day 15 Other Effector; Mus musculus; ATAC-seq
library_construction_protocol: ATAC-Seq was performed using a previously described optimization of the original protocol of Corces et al. (Nature Genetics, 2017). T helper cells were sorted using a BD FACSAria Fusion from the lungs (indlucing airways) of mice 15 days after sensitization and challenge with HDM. Four populations were sorted – Th2 cells (CD3+CD4+CD44+ST2+CD30L+), Non-Th2 memory cells (CD3+CD4+CD44+ST2-CD30L+), Treg (CD3+CD4+CD25hiCD30L-) and naïve T helper cells (from mLN, CD3+CD4+CD44loCD62Lhi). Three biological replicates were sorted for each population. In total, 1000 of each T helper cell subset was purified from the combined lung and airway tissue. Cells were sorted directly into 25 µl of Tn5 transposase buffer (TD Buffer, 0.02 % Digitonin, Nextera DNA Library Prep Kit, Illumina). Immediately after sorting, 2.5 µl Tn5 transposase was added to each well on ice and mixed thoroughly by pipetting. The plate was next sealed and incubated at 37 oC for 30 minutes on a standard thermocycler. The reaction was stopped by adding 300 µl ERC buffer (QIAGEN MinElute Reaction Clean up kit) and fragmented DNA was isolated by column purification (QIAGEN, MinElute). Total fragmented DNA was subjected to PCR amplification (NEBNext PCR master mix, New England Biolabs) with illumina adapters (indexes 1-12) for 16 cycles (for indexes see Corces et al.). Final libraries were size-selected with Ampure XP magnetic beads (Beckman Coulter) and pooled for sequencing on a Hiseq2500 on high output mode with paired-end 2x 125 reads (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM3831242