Antigen

Antigen Class

ATAC-Seq

Antigen

ATAC-Seq

Cell type

Cell type Class

Blood

Cell type

Macrophages

MeSH Description

The relatively long-lived phagocytic cell of mammalian tissues that are derived from blood MONOCYTES. Main types are PERITONEAL MACROPHAGES; ALVEOLAR MACROPHAGES; HISTIOCYTES; KUPFFER CELLS of the liver; and OSTEOCLASTS. They may further differentiate within chronic inflammatory lesions to EPITHELIOID CELLS or may fuse to form FOREIGN BODY GIANT CELLS or LANGHANS GIANT CELLS. (from The Dictionary of Cell Biology, Lackie and Dow, 3rd ed.)

Sample

source_name

alveolar macrophages

strain

DBA2/J

Sequenced DNA Library

library_strategy

ATAC-seq

library_source

GENOMIC

library_selection

other

library_construction_protocol

Lungs were incubated at 37oC for 15 minutes while on a rotator. Red blood cells were lysed using RBC lysis buffer (eBioscience) for a 5 minute incubation. The obtained cell suspension was filtered using a 70 micron filter, washed and cells were resuspended in PBS. Cells were then incubated with anti-Fc blocking mAb (anti CD16/32, clone 93, BioLegend) and viability dye Zombie Aqua (Biolegend) for 10 min. After this, cells where stained with an antibody cocktail containing APC Cy7-conjugated CD45, PE-conjugated F4/80, BV605-conjugated CD11c, v450-conjugated CD11b, PE Cy7-conjugated CD64, AF700-conjufgated Ly6c, FITC-conjugated Ly6g, AF647-conjugated SiglecF mAb for 30 minutes. All labeled cells were passed through a FACS Aria II flow cytometer (BD Biosciences) and after proper gating 4 subsets of lung MP were sorted RNA-seq: PolyA enriched mRNA was fragmented, in 2x Superscript III first-strand buffer with 10mM DTT (Invitrogen), by incubation at 94°C for 9 minutes, then immediately chilled on ice before the next step. The 10 µL of fragmented mRNA, 0.5 µL of Random primer (Invitrogen), 0.5 µL of Oligo dT primer (Invitrogen), 0.5 µL of SUPERase-In (Ambion), 1 µL of dNTPs (10 mM) and 1 µL of DTT (10 mM) were heated at 50°C for three minutes. At the end of incubation, 5.8 µL of water, 1 µL of DTT (100 mM), 0.1 µL Actinomycin D (2 µg/µL), 0.2 µL of 1% Tween-20 (Sigma) and 0.2 µL of Superscript III (Invitrogen) were added and incubated in a PCR machine using the following conditions: 25°C for 10 minutes, 50°C for 50 minutes, and a 4°C hold. The product was then purified with RNAClean XP beads according to manufacture's instruction and eluted with 10 µL nuclease-free water. The RNA/cDNA double-stranded hybrid was then added to 1.5 µL of Blue Buffer (Enzymatics), 1.1 µL of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 µL of RNAse H (5 U/µL), 1.05 µL of water, 1 µL of DNA polymerase I (Enzymatics) and 0.15 µL of 1% Tween-20. The mixture was incubated at 16°C for 1 hour. The resulting dUTP-marked dsDNA was purified using 28 µL of Sera-Mag Speedbeads (Thermo Fisher Scientific), diluted with 20% PEG8000, 2.5M NaCl to final of 13% PEG, eluted with 40 µL EB buffer (10 mM Tris-Cl, pH 8.5) and frozen -80°C. The purified dsDNA (40 µL) underwent end repair by blunting, A-tailing and adapter ligation as previously described44 using barcoded adapters (NextFlex, Bioo Scientific). Libraries were PCR-amplified for 14-16 cycles, size selected using 10% PAGE/TBE gels, eluted and quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), ATAC-seq: Sorted cells in FACS Buffer were pelleted by centrifugation at 400 RCF for 6 minutes at 4oC in a pre-cooled fixed-angle microfuge. Supernatant was carefully removed and cell pellet was resuspend in 47.5 ul lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), 2.5ul of TDE1 (Cat# FC-121-1030, Illumina) by pipetting. Transposition reactions were incubated at 37°C for 30 minutes. Transposed DNA was purified using a Zymo ChIP DNA Clean & Concentrator kit (Cat# D5205) and purified DNA was eluted in 10 ul elution buffer (10 mM Tris-HCl, pH 8). Transposed fragments were amplified and purified as described previously (Buenostro et al) with modified primers.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

11389640

Reads aligned (%)

44.8

Duplicates removed (%)

58.9

Number of peaks

15279 (qval < 1E-05)

mm9

Number of total reads

11389640

Reads aligned (%)

44.8

Duplicates removed (%)

58.9

Number of peaks

15267 (qval < 1E-05)