organism: Homo sapiens
description: Protocols: Peripheral blood (PB) and bone marrow (BM) samples were harvested in EDTA containing tubes and stored at room temperature. Samples were processed within 2h from collection. Mononuclear cells and granulocytes were separated by density centrifugation over a Lymphoprep (Stemcell Technologies) gradient. Granulocyte enriched fraction was further purified over a Hetasep gradient. Normal density neutrophils (NDNs) were isolated from total granulocytes by magnetic bead sorting using Neutrophil Isolation kit (StemCell Technologies). Monocytes and low density neutrophils (LDNs) were isolated by magnetic bead sorting (CD14 Microbeads or CD15 Microbeads Miltenyi, respectively) or by fluorescence activated cell sorting (FACS). We sorted monocytes as CD3-CD56-CD19-CD34-(Lin-)CD33+CD15-CD14+ cells and LDN as (Lin-)CD33+CD14-CD15+CD193- cells. BM neutrophil developmental intermediates were isolated by FACS sorting and identified as (Lin-)CD14-CD33+CD15+CD193- cells and further fractionated into: BM1 CD11b-CD16- cells, BM2 CD11b+CD16- cells, BM3 CD11b+CD16int, and BM 4 CD11b+CD16hiCD10+ cells. Cells were sorted at MoFlo XDP (Beckman Coulter) or FACSAria Fusion (Beckton Dickinson) cell sorters. We extracted total RNA using the ReliaPrep RNA Cell Miniprep System (Promega). We quantified RNA concentration using Qubit RNA HS Assay Kit using Qubit 3.0 and we evaluated RNA integrity with Agilent RNA 6000 Pico Kit using Bionalyzer (Agilent). RNA-Seq libraries were generated using the Smart-seq2 method (Picelli et al., 2014) starting from 0.5 ng of RNA. Retro-transcription was performed using SuperScript II Reverse Transcriptase, cDNA was PCR-amplified (18 cycles) with KAPA HiFi HotStart and purified with AMPure XP beads. After purification, we determined cDNA concentration using Qubit dsDNA HS Assay Kit at Qubit 3.0 and we assessed size distribution at Agilent 4200 TapeStation system. We performed the tagmentation reaction starting from 0.5 ng of cDNA for 30 minutes at 55°C and we performed enrichment PCR using 12 cycles. Libraries were purified with AMPure XP beads, quantified using Qubit 3.0, assessed for fragment size distribution on an Agilent 4200 TapeStation system.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-09-09
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-09-09
attributes.tag: External Id
attributes.value: SAMEA11307688
attributes.tag: INSDC center alias
attributes.value: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute
attributes.tag: INSDC center name
attributes.value: San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute
attributes.tag: INSDC first public
attributes.value: 2022-09-09T12:17:12Z
attributes.tag: INSDC last update
attributes.value: 2022-09-09T12:17:12Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11190:Sample 126
attributes.tag: age
attributes.value: 36
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell isolation strategy
attributes.value: CD15 Microbeads
attributes.tag: cell_type
attributes.value: low density neutrophil
attributes.tag: clinical history
attributes.value: G-CSF
attributes.tag: common name
attributes.value: human
attributes.tag: immunophenotype
attributes.value: (Lin-)CD33+CD14-CD15+CD193-
attributes.tag: individual
attributes.value: UPN1
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-11190:Sample 126
attributes.tag: sex
attributes.value: female