Instrument: NextSeq 550
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: RNA-seq: Total RNA was prepared from approximately 100,000-500,000 cells using an RNeasy Plus Micro Kit (Qiagen). RNA integrity numbers were determined using a TapeStation 2200 (Agilent) and all samples had RIN numbers above 9.5. ChIP-seq: Cells were chemically cross-linked by 1% formaldehyde. Samples were sonicated and protein-DNA complexes were isolated with antibody. ATAC-seq: ATAC-seq was performed as previously described with minor modifications (Buenrostro et al., Nature Methods, 2013). Fifty thousand cells from FACS or from cultur were pelleted at 550 x g and washed with 1 mL 1x PBS followed by lysis with 50 μL lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Accessible chromatin was tagmented with Tn5 transposase (FC-121-1030; Illumina) for 45-75 minutes depending on the cell-type in a 37°C waterbath. scATAC-seq: Single cell ATAC-seq was performed as previously described with minor modifications (Buenrostro et al., Nature, 2015) using the C1 Single-Cell Auto Prep System with the C1 Open App program (Fluidigm). Cells were FACS sorted and stained with mammalian LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen) for 10 minutes on ice at a final concentration of 5 μM Ethidium homodimer-1 and 5 μM Calcein AM in 1x PBS. After staining, cells were diluted in RPMI-1640+1% FBS to a concentration of 400,000 cells per mL. C1 Cell Suspension Reagent (Fluidigm) was added to a final concentration of 20%. Cell capture was verified by light microscope (for Capture 1). Brightfield and fluorescent images of each capture site was taken with a Leica DMi8 (for Captures 2 and 3). The Lysis/Tagmention step in the C1 protocol was lengthened to a duration of 60 minutes using the Open App software (Fluidigm). RNA-seq: RNA-seq libraries were prepared using the SMARTer High-Input Strand-Specific Total RNA-seq for Illumina kit (Clontech) and were single-end sequenced for 75 bp. ChIP-seq: ChIP DNA was approximately 300 bp in length. Libraries were prepared using Ultra DNA Library Prep Kit (NEB) and single-end sequenced for 75 bp. ATAC-seq: Tagmented DNA was amplified for 12 cycles of PCR, purified, and the prepared libraries were paired-end sequenced (38bp+37bp). scATAC-seq: After single cell ATAC-seq chemistry was performed on the Fluidigm C1, tagmented DNA from single cell libraries were harvested and amplified for 14 PCR cycles. Libraries were pooled and paired-end sequenced (38bp+37bp).