Antigen

Antigen Class

DNase-seq

Antigen

DNase-Seq

Cell type

Cell type Class

Blood

Cell type

Erythroid Cells

MeSH Description

The series of cells in the red blood cell lineage at various stages of differentiation.

Sample

source_name

Erythroid cells

tissue

blood

genes analysed

Genome-wide

Sequenced DNA Library

library_strategy

DNase-Hypersensitivity

library_source

GENOMIC

library_selection

DNase

library_construction_protocol

Mouse: A single cell suspension was made by gently dissociating the PHZ spleens and passing it through a 70um filter. Cells were washed in cold PBS, and counted. 50million cells were used for the DNase-Seq protocol. Nuclei were isolated by lysing the cells and digested with increasing concentrations of DNaseI to isolate open chromatin fragments as previously published (Hosseini, 2014). Human: Primary erythroid stem cell progenitors were isolated from peripheral blood, using CD34 coupled magnetic beads. Cells were expanded for 7days in low Epo conditions (0.5 IU/ml)) and then transfered for differentiation in high Epo (3.0 IU/ml) medium. On day 13, cells were washed in cold PBS, and counted. 50million cells were used for the DNase-Seq protocol. Nuclei were isolated by lysing the cells and digested with increasing concentrations of DNaseI to isolate open chromatin fragments as previously published (Hosseini, 2014, DOI 10.1371/journal.pgen.1003570). The DNase-Seq protocol was perfromed as previously published (Hosseini 2014). DNA was purified using a phenol choroform extraction and the optimal disgests were selected for library preparation using NEB Library Preparation kit. Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and TruSeq oligos. A size selection step for small fragments was performed. DNase-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced on Illumina platform using: 150bp paired end reads (MiSeq), 75bp paired end reads (HiSeq) or 40/75 bp (NextSeq) paired end reads. For the background libraries, 100ng of genomic DNA was incubated with DNase for 3 minutes and then libraries were created to test sequence for sequence bias. The strategy was to isolate open chromatin fragments specifically as they underly regulatory genomic regions. These are short stretches of DNA, with low abundance in the genome and thus require to be processed efficiently. The fragments can then be multiplexed and sequenced.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

160797499

Reads aligned (%)

70.7

Duplicates removed (%)

8.1

Number of peaks

3280 (qval < 1E-05)

mm9

Number of total reads

160797499

Reads aligned (%)

70.5

Duplicates removed (%)

8.5

Number of peaks

4191 (qval < 1E-05)