title: Actin dependent astrocytic infiltration is a key step for axon defasciculation during remodeling

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: A strocytes are essential for synapse formation, maturation and plasticity, yet their function during developmental neuronal remodeling is largely unknown To identify astrocytic molecules required for axon pruning of mushroom body ( γ neurons in Drosophi la we profile d the expression of astrocytes before ( and after ( remodeling. F ocus ing on genes enriched in larval astrocytes we identified 12 genes that are required in astrocytes, for axon pruning including the F actin regulat ors Arpc1 and form3 Interestingly, perturbing astrocytic actin dynamics did not affect their gross morphology migration or TGF β secretion In contrast, actin dynamics was required for astrocyte infiltration into the axon bundle at the onset of pruning. Remarkably, decreasing axonal adhesion facilitated the infiltration of Arpc1 KD astrocytes, and suppressed the pruning defect driven by the astrocytic perturbation Together, our findings suggest that actin dependent astrocytic infiltration is a key step in axon pruning thus promoting our understanding of neuron glia interactions during remodeling

overall_design: Transcriptional profiling of drosophila astrocytes before (larva) and after (adult) neuronal remodeling.

first: Neta

last: Marmor-Kollet

first: Hadas

last: Keren-Shaul

first: Eyal

last: David

first: Oren

last: Schuldiner


title: Genome-wide chromatin profiles of PBRM1 [RNA-Seq]

status: Public on Feb 13 2023

city: Singapore

first: Xiaosai

last: Yao

address: 60 Biopolis Street, Genome, #02-01

country: Singapore

institute: Genome Institute of Singapore

type: Expression profiling by high throughput sequencing

summary: PBRM1 is an accessory subunit of the PBAF subclass of the SWI/SNF chromatin remodeler. The inactivation of PBRM1 is the second most frequent mutational event in kidney tumorigenesis. Here we show that in VHL-deficient ccRCC tumors, PBRM1 loss results in an altered PBAF complex that retains the association between SMARCA4 and ARID2 but disengages BRD7 from SMARCA4. The PBRM1-deficient PBAF complexes redistribute from promoter proxy regions to distal enhancer regions. The ATPase function of SMARCA4 enhances the recruitment of nuclear factor RELA to aberrant sites and promotes NF-κB activity. Proteasome inhibitor bortezomib reverses NF-κB activation by reducing RELA binding at regions bound by PBRM1-deficient PBAF and delays PBRM1-deficient tumor growth. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumorigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.

overall_design: Comparison of transcriptomic changes with and without PBRM1

first: Bin

middle: T

last: Teh


title: Genome-wide RNAseq of hippocampi from single time point (2 hours post 0.75 mg/mL corticosterone injection) in adrenalectomized rats

status: Public on Feb 12 2023

city: Irvine

first: Matthew

last: Birnie

address: Health Sciences Road

country: USA

institute: University of California-Irvine

summary: Disrupted circadian activity is associated with many neuropsychiatric disorders. A major coordinator of circadian biological systems is adrenal glucocorticoid secretion which exhibits a pronounced pre-awakening peak that regulates metabolic, immune, and cardiovascular processes, as well as mood and cognitive function. Loss of this circadian rhythm during corticosteroid therapy is often associated with memory impairment. Surprisingly the mechanisms that underlie this deficit are not understood. In this study, in rats, we report that circadian regulation of the hippocampal transcriptome integrates crucial functional networks that link corticosteroid-inducible gene regulation to synaptic plasticity processes via an intra-hippocampal circadian transcriptional clock. Further, these circadian hippocampal functions were significantly impacted by corticosteroid treatment delivered in a five day oral dosing treatment protocol. Rhythmic expression of the hippocampal transcriptome, as well as the circadian regulation of synaptic plasticity were misaligned with the natural light/dark circadian entraining cues, resulting in memory impairment in hippocampal-dependent behavior. These findings provide mechanistic insights into how the transcriptional clock machinery within the hippocampus is influenced by corticosteroid exposure, leading to adverse effects on critical hippocampal functions, as well as identifying a molecular basis for memory deficits in patients treated with long-acting synthetic corticosteroids.

overall_design: Hippocampus RNA expression from adult adrenalectomized vehicle and corticosterone treated rats at zeitgeiber (ZT) 2

first: Stafford

middle: L

last: Lightman

first: Becky

last: Conway-Campbell


title: RNA-sequencing of NR2F2-isoform 2 melanoma cellular models

city: New York

first: Igor

last: Dolgalev

state: New York

address: 550 1st Ave

institute: NYU Grossman School of Medicine

summary: Melanoma is a highly metastatic tumor type responsible of the large majority of the skin cancer-related deaths. Melanoma cells recapitulate the migratory and invasive nature of neural crest stem cells from which melanocytes arise. However, the mechanisms by which these developmental programs confer melanoma cells with more aggressive properties are not understood. Here we provide evidence for an epigenetically regulated developmental program that is aberrantly re-activated by melanoma cells to aid in the metastatic process. We reasoned that studying molecular changes occurring during the ontogeny of melanocytes from progenitor neural crest cells (NCC), and identifying developmental programs retained or re-gained by melanoma cells, could reveal crucial processes that modulate melanoma aggressiveness. Here we describe a novel mechanism that controls the activity of a transcriptional regulator of human neural crest, the Nuclear Receptor Subfamily 2 Group F, Member 2 (NR2F2). We find that highly localized DNA methylation acts as an on/off switch that controls the expression of a truncated NR2F2 isoform (NR2F2-Iso2) from an alternative transcription start site during NCC to melanocyte differentiation. We show that melanoma cells co-opt this developmental program by decreased DNA methylation and re-expression of NR2F2-Iso2 to promote melanoma metastasis. NR2F2-Iso2 regulates the transcriptional activity of the full length NR2F2 isoform 1 by impairing its binding to chromatin, which results in altered expression of NCC and differentiation genes. Our data demonstrate that epigenetic reactivation of NR2F2 isoform 2 promotes melanoma metastasis, which could be targeted for therapeutic purposes.

overall_design: NR2F2-isoform 2 loss-of-function (shRNA) and gain-of-function (ectopic expression) cellular models were generated in 4L and MeWo melanoma cell lines, respectively.

first: Veronica

last: Davalos

first: Eva

last: Hernando


title: NR2F2 ChIP-sequencing of melanoma cell lines

type: Genome binding/occupancy profiling by high throughput sequencing

overall_design: NR2F2-isoform 2 loss-of-function (shRNA) cellular models were generated in 4L melanoma cell line.


title: Strategies for optimizing CITE-seq for human islets and other tissues

city: Vancouver

first: Megan

last: Levings

state: BC

address: 950 West 28th Avenue

country: Canada

institute: University of British Columbia

summary: Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor- matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITEseq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITEseq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

overall_design: We performed CITE-Seq on enzymatically digested, CD45-enriched, flow-sorted human islets to assess the ability of CITE-Seq oligo-antibodies to bind to target cells. We developed a panel of 61 CITE-Seq oligo-antibodies to select for key lineage and phenotypic immune cell markers, and evaluated how the enzymatic digestion process affected detection of these key cell surface proteins. We repeated this enzymatic digestion on mechanically dissociated human splenocytes and used the same panel of 61 CITE-Seq olig-antibodies to compare immune cell composition to that of islet-resident CD45+ cells.

first: Sarah

middle: J

last: Colpitts

middle: A

last: Budd

first: Mahdis

last: Monajemi

first: Kyle

last: Reid

first: Julia

middle: M

last: Murphy

first: Sabine

last: Ivison

first: C

middle: B

last: Verchere

middle: K

middle: Q

last: Crome


title: Murine Endogenous Retrovirus MERVL is essential for the transition from Totipotency to Pluripotency during Preimplantation Development

city: Shinjuku-ku

first: Akihiko

last: Sakashita

state: Tokyo

address: 35 Shinanomachi

department: Department of Molecular Biology,

country: Japan

institute: Keio University School of Medicine

summary: Zygotic genome activation (ZGA) is a critical post-fertilization step that promotes totipotency and allows different cell fates to emerge in the developing embryo. MERVL, murine endogenous retrovirus-L, is transiently upregulated at the 2-cell stage around the time of ZGA. Although MERVL expression is widely used as a marker of totipotency, the role of this retrotransposon in mouse embryogenesis remains elusive. Here, we develop methods for consistent knockdown (KD) and inactivation of interspersed copies of MERVL and show that MERVL transcripts, but not encoded retroviral proteins and their long terminal repeat (LTR) promoter that drives chimeric transcripts with host genes, is essential for accurate regulation of the host transcriptome and chromatin state during preimplantation development. Both KD and CRISPRi-based repression of MERVL result in embryonic lethality due to defects in differentiation and genomic stability. Furthermore, transcriptome and epigenome analysis revealed that loss of MERVL transcript led to retain an accessible chromatin state at, and aberrant expression of, a subset of 2-cell specific genes at mid-preimplantation stages. Taken together, our results suggest a model in which an endogenous retrovirus plays a critical role in regulating host cell fate potential.

overall_design:
RNA-seq and ATAC-seq analyses of 2-cell, 4-cell and 8-cell stage embryos from control and MERVL-KD mouse preimplantation embryos.
RNA-seq analysis of 2-cell, 4-cell and 8-cell stage embryos upon CRISPRi-mediated repression of MERVL.

first: Tomohiro

last: Kitano

first: Haruhiko

last: Siomi


title: A virus-borne DNA damage signaling pathway controls the lysogeny-induction switch of a group of temperate pleolipoviruses

status: Public on Feb 11 2023

city: wuhan

first: chen

last: zhao

state: huibei

address: luojiashan

department: college of life sciences

country: China

institute: wuhan university

summary: To determine the difference in gene expression of SNJ2 genes with and without MMC treatment, Natrinema sp. J7-1 was incubated to middle exponential phase (OD600=0.6-0.8) in Halo-2 medium and treated with MMC

overall_design: Cell samples were then collected at 0, 1, 4 and 9h after MMC treatment. RNA extraction and RNAseq was performed

first: Zhao

last: Chen

first: Ying

last: Liu

first: Shishen

last: Du

first: Xiangdong


title: Genome-wide RNAseq from multi time point (every 4 hours) rat hippocampi with vehicle and methylprednisolone (MPL) treatment

first: Matthew

last: Birnie

overall_design: Hippocampus RNA expression from adult CTL and methylprednisolone treated rats at zeitgeiber (ZT) 2,6,10,14,18,22

first: Stafford

last: Lightman

first: Becky

last: Conway-Campbell


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity [Barcode_decay_in_bulk_HA1ER]

city: Glasgow

first: Maximiliano

last: PORTAL

address: Garscube Estate. Switchback road

country: United Kingdom

institute: Cancer Research UK Scotland Institute - The University of Glasgow

summary: Cell plasticity is a core biological process underlying a myriad of molecular and cellular events taking place throughout organismal development and evolution. It has been postulated that cellular systems thrive to balance the organization of meta-stable states underlying this phenomenon, thus maintaining a degree of populational homeostasis compatible with an ever-changing environment and, thus, life. Notably, albeit circumstantial evidence has been gathered in favour of the latter conceptual framework, a direct observation of meta-state dynamics and the biological consequences of such a process in generating non-genetic clonal diversity and divergent phenotypic output remains largely unexplored. To fill this void, we developed a lineage-tracing technology termed Barcode-decay Lineage Tracing-Seq. BdLT-Seq is based on episome-encoded molecular identifiers that, supported by the dynamic decay of the tracing information upon cell division, ascribe directionality to a cell lineage tree in a time-resolved manner whilst directly coupling non-genetic molecular features to phenotypes in comparable genomic landscapes. Herein we show that cell transcriptome states are both inherited and dynamically reshaped following constrained rules encoded within the cell lineage, leading to intra-clonal non-genetic diversity in basal growth conditions and while adjusting populational phenotypic output upon oncogene activation and throughout the process of reversible resistance to therapeutic cues.

overall_design: Sequencing of BdLT-seq barcodes "in bulk" in HA1ER F12 clone. To monitor a barcode "decay" over time several time points were analysed - "0 days", "5 days", "10 days" and "20 days".

first: Yelyzaveta

last: Shlyakhtina

first: Bianca

last: Blochl

first: Maximilliano

last: Portal


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity [scRNAseq_HA1ER_clone_F12_1F8_1C9]

overall_design: Clonal populations of HA1ER cell line - F12, 1F8 and 1C9 were analyzed by scRNA-seq. Three samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies: HA1ER F12 - CTCCTCTGCAATTAC, HA1ER 1F8 - CAGTAGTCACGGTCA, HA1ER 1C9 - ATTGACCCGCGTTAG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity [scRNAseq_BdLTseq_HA1E_E7_clone_RAS]

overall_design: BdLT-seq in HA1E E7 clone expressing six RAS variants (HRAS-Q61K, HRAS-G12C, KRAS-G12V, KRAS-E62K, NRAS-Q61R, NRAS-Y64D). Lineage tracing was performed using BdLT-seq in HA1E-E7-RAS cells from Day 0 to Day 5, standard growing conditions. Two samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1E E7_ RAS_0days - TGTCTTTCCTGCCAG and CTCCTCTGCAATTAC, HA1E E7_ RAS_5days_1 - CAGTAGTCACGGTCA and ATTGACCCGCGTTAG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity  [scRNAseq_BdLTseq_HA1E_E7_clone]

overall_design: scRNA-seq and BdLT-seq in HA1E cell line E7 clone. Lineage tracing using BdLT-seq was performed from Day 0 to Day 5 in standard culture conditions. Two samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1E E7_0days - CAGTAGTCACGGTCA, HA1E E7_5days - ATTGACCCGCGTTAG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity  [scRNAseq_BdLTseq_HA1ER_F12_clone_3]

overall_design: BdLT-seq in HA1ER F12 clone. Lineage tracing using BdLT-seq was performed from Day 0 to Day 5 in standard culture conditions. Four samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1ER F12_0days - TGTCTTTCCTGCCAG and CTCCTCTGCAATTAC, HA1ER F12_5days - CAGTAGTCACGGTCA and ATTGACCCGCGTTAG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity  [scRNAseq_BdLTseq_HA1ER_F12_clone_TRAIL]

overall_design: scRNA-seq and BdLT-seq in HA1ER clones F12, 1F8 and 1C9. Lineage tracing was performed using BdLT-seq in HA1ER F12, HA1ER 1F8 and HA1ER 1C9 cells resistant to TRAIL-induced apoptosis following TRAIL withdrawal (3 days). Six samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1ER F12_TRAIL_resistant - AAGTATCGTTTCGCA, HA1ER F12_TRAIL_withdrawal - GGTTGCCAGATGTCA, HA1ER 1F8_TRAIL_resistant - TGTCTTTCCTGCCAG, HA1ER 1F8_TRAIL_withdrawal - CTCCTCTGCAATTAC, HA1ER 1C9_TRAIL_resistant - CAGTAGTCACGGTCA, HA1ER 1C9_TRAIL_withdrawal - ATTGACCCGCGTTAG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity  [scRNAseq_BdLTseq_HA1ER_F12_clone_1]

overall_design: BdLT-seq in HA1ER F12 clone. Lineage tracing using BdLT-seq was performed from Day 0 to Day 7 to Day 14 in standard culture conditions. Three samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1ER F12_0days - GTCAACTCTTTAGCG, HA1ER F12_7days - TGATGGCCTATTGGG, HA1ER F12_14days - TTCCGCCTCTCTTTG.


title: Barcode decay Lineage Tracing, BdLT-Seq, unravels lineage-linked transcriptome plasticity [scRNAseq_BdLTseq_HA1ER_F12_clone_2]

overall_design: BdLT-seq in HA1ER F12 clone. Lineage tracing using BdLT-seq was performed from Day 0 to Day 5 in standard culture conditions. Two samples were multiplexed on a single Chromium 10x run (single lane) using hashtag antibodies - HA1ER F12_0days - GTCAACTCTTTAGCG and TTCCGCCTCTCTTTG, HA1ER F12_5days - TGATGGCCTATTGGG and AGTAAGTTCAGCGTA.


title: PRRC2 proteins are translation factors that promote leaky scanning

city: Heidelberg

first: Jonathan

last: Bohlen

address: Im Neuenheimer Feld 581

country: Germany

institute: DKFZ

summary: Most animal mRNAs contain upstream Open Reading Frames (uORFs). These uORFs represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5’ end and then scan for ORFs in a 5’-to-3’ fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important post-transcriptional mechanism affecting gene expression. Few molecular factors regulating or facilitating this process are known. Here we identify the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C as translation initiation factors. We find that they bind other eukaryotic translation initiation factors and preinitiation complexes and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote translation of mRNAs containing uORFs by facilitating leaky scanning past uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.

overall_design: Selective and total 40S and 80S ribosome footprinting in control versus PRRC2A+B+C knockdown condition. Selective Ribosome footprinting for PRRC2C. Also RNA-seq.

first: Mykola

last: Roiuk

first: Aurelio

last: Teleman


title: The effect of ionomycin-induced oocyte activation on multiple morphological abnormalities of the sperm flagella

city: Fuzhou

first: Zhiren

state: Fujian

address: 20 Chazhong Road

institute: the First Affiliated Hospital of Fujian Medical University

summary: Artificial oocyte activation (AOA) is considered an effective method to improve clinical outcomes in patients with some forms of male factor infertility and does not increase the risk of birth defects. However, the effects of AOA on patients with multiple morphological abnormalities of the sperm flagella (MMAF) caused by a DNAH1 mutation are still unknown. To explore the effects, our study analyzed a case with MMAF due to DNAH1 homozygous mutation who underwent testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI). The case had 28 MII oocytes. The 28 oocytes were divided randomly and equally into AOA and non-AOA groups. Ionomycin was used for AOA. We compared the clinical outcomes of two groups and selected three blastulation failure embryos from each group for transcriptome analysis. Differentially expressed genes (DEGs) were determined with an adjusted P-value < 0.05 and a |log2-fold change| ≥ 1. The comparison of clinical outcomes showed that the two pronuclei (2PN) rate and grade 1–2 embryo rate at day 3 were not significantly different between the two groups. Transcriptome analyses of blastulation failure embryos showed that the use of AOA had potential risks of chromosome structure defects, transcriptional regulation defects, and epigenetic defects. In conclusion, when the case with MMAF due to DNAH1 mutation underwent TESE-ICSI, ionomycin-induced oocyte activation could not improve the clinical outcomes and introduced the risks of chromosome structure defect, transcriptional regulation defect, and epigenetic defect.

overall_design: Our study analyzed a case with MMAF due to DNAH1 homozygous mutation who underwent testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI). The case had 28 MII oocytes. The 28 oocytes were divided randomly and equally into AOA and non-AOA groups. Ionomycin was used for AOA. We compared the clinical outcomes of two groups and selected three blastulation failure embryos from each group for transcriptome analysis.

first: Mingting

last: Jiang

first: Qicai


title: Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF

status: Public on Feb 10 2023

city: Tomsk

first: Irina

last: Larionova

state: Tomsk oblast

address: Lenin Ave, 36, Tomsk, Tomsk Oblast, Russia

country: Russia

institute: Tomsk State University

summary: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated, that TAMs scavenger receptor stabilin-1 is responsible for the clearance of EGF, a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on SYT11, confirmed in patients with breast cancer. Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

overall_design: Human monocytes were obtained from the German Red Cross Blood Service Baden-Württemberg– Hessen (Mannheim, Germany). Three donors of German cohort were used for preparation of ex vivo TAMs and whole-transcriptome sequencing. Monocytes were isolated from the buffy coats of healthy donors by density gradients followed by positive magnetic selection using CD14+ MACS beads (no. 130-050-201, Miltenyi Biotech, Germany), resulting to 90–98% monocyte purity as confirmed by flow cytometry. Monocytes were cultured at the concentration of 106 cells/ml in serum-free X-VIVO medium (Lonza, Germany) supplemented with 10 ng/ml of macrophage colony-stimulating factor (M-CSF) (no. 300-25, Peprotech, Germany), 10−8 M of dexamethasone (no. D2915, Sigma-Aldrich, Germany) and 10 ng/ml of IL-4 (no. 200-04, Peprotech, Germany). Supernatants from MCF-7 cells were added to freshly isolated human primary monocytes at a final volume of 20% of a cultivation medium. Monocytes were differentiated to TAMs for 6 days in the presence of 7.5% CO2. Cisplatin (Cisplatin-Teva, Teva Pharmaceutical Industries, Ltd. L01XA01 injection solution) was used for chemotherapeutic treatment of TAMs in vitro. For ex vivo TAMs, cisplatin was used at the selected concentrations (20 μM, 40 μM, and 80 μM) to determine the optimal concentration for further analysis. The 20 μМ concentration of cisplatin was used for further analysis. Cisplatin was added on day 6 of macrophages differentiation and TAMs were incubated with cisplatin for 3 days. Whole-transcriptome sequencing was performed on 3 untreated TAMs and 3 TAMs treated with cisplatin. Prepared libraries were then pooled and sequenced on an Illumina NextSeq 500 instrument (Illumina, USA) with NextSeq 500/550 High-Output v2.5 Kit (75 cycles) (cat #20024906).

first: Artem

last: KIselev

first: Elena

last: Kazakova

first: Tengfei

first: Marina

last: Patysheva

first: Pavel

last: Iamshchikov

first: Dieuwertje

last: Mossel

first: Vladimir

last: Riabov

first: Militsa

last: Rakina

first: Alexey

last: Sergushichev

first: Natalia

last: Bezgodova

first: Sergei

last: Vtorushin

first: Nikolai

last: Litviakov

first: Evgeny

last: Denisov

first: Philipp

last: Koshkin

first: Denis

last: Pyankov

first: Matvei

last: Tsyganov

last: Ibragimova

first: Nadezhda

last: Cherdyntseva

first: Julia

last: Kzhyshkowska


title: ChIP-Seq based CTCF binding profiling of naïve B-cells from CTCFfl/fl; CD19-Creki/+ or CTCFfl/+; CD19-Creki/+ mice.

city: Madrid

first: Manuel

last: Gomez

state: Madrid

address: Melchor Fernández Almagro, 3

country: Spain

institute: CNIC

summary: The goal of this study was to compare the CTCF binding expression profile, using ChIP-seq technology, of naïve B-cells  from CTCFfl/fl; CD19-Creki/+ or CTCFfl/+; CD19-Creki/+ mice.

overall_design:
Naïve B cells, isolated from the spleen of CTCFfl/fl; CD19-Creki/+ or CTCFfl/+; CD19-Creki/+ mice, were used to extract genomic DNA with the aim of studying CTCF binding site occupancy using ChIP-Seq.

C* samples have genotype CTCF fl / +; CD19-Cre ki/+; they are heterozygous for the Ctcf-floxed allele.
F* samples have genotype CTCF fl / fl; CD19-Cre ki/+; they are homozygous for the Ctcf-floxed allele.
CTCF Antibody (Diagenode, C15410210-10, lot A2359-00234P)

first: A

last: Ramiro


title: Profiling the genomic binding of DAXX in C9HRE ALS/FTD patient cells

city: Baltimore

first: Yang

last: Liu

state: MD

address: 615 N. Wolfe Street

department: Biochemistry and Molecular Biology

institute: Johns Hopkins University

summary: Using ChIP-seq to profile the genomic binding of DAXX in control and C9HRE ALS/FTD patient B lymphocytes.

overall_design: Tow pairs of control and patient B lymphocytes were used.

first: Jiou

last: Wang


title: To profile the chromatin structural changes reprogrammed by the liquid-liquid phase separation of DAXX [HiChIP]

summary: DAXX is an epigenetic regulator and chromatin chaperon protein. This experiment aims to investigate whether DAXX's phase separation causes changes in chromatin structures.

overall_design: DAXX  was fused with a Cry2 domain which enables DAXX to form condensates upon blue light illumination. HiChIP was employed to profile the long-range chramatin interactions in HEK293 cells expressing DAXX-mCherry-Cry2 with or without blue light illumination.


title: Denervated mouse CA1 pyramidal neurons express homeostatic synaptic plasticity following entorhinal cortex lesion

city: Hannover

first: Maximilian

last: Lenz

address: Carl-Neuberg-Straße 1

department: Institute of Neuroanatomy and Cell Biology

institute: Hannover Medical School

summary: Structural, functional, and molecular reorganization of denervated neural networks is often observed in neurological conditions. The loss of input is accompanied by homeostatic synaptic adaptations, which can affect the reorganization of denervated networks. However, a major challenge of denervation-induced homeostatic plasticity operating in complex neural networks is the specialization of neuronal inputs. Therefore, it remains unclear whether neurons respond similarly to the loss of distinct inputs. Here, we used in vitro entorhinal cortex lesion (ECL) and Schaffer collateral lesion (SCL) in mouse organotypic entorhino-hippocampal tissue cultures of either sex, and studied denervation-induced plasticity of CA1 pyramidal neurons. We observed accumulation of microglia, degeneration of presynaptic buttons and a reduction in dendritic spine numbers in the denervated layers three days after SCL and ECL, respectively. Transcriptome analysis of the CA1 region showed complex changes in differential gene expression following SCL and ECL compared to non-lesioned controls. An enrichment of differentially expressed synapse-related genes was observed specifically after ECL. Consistent with this finding, denervation-induced homeostatic plasticity of excitatory synapses was observed three days after ECL but not after SCL. Chemogenetic silencing of the EC but not CA3 confirmed the pathway-specific induction of homeostatic synaptic plasticity in CA1. Moreover, increased RNA oxidation was observed after SCL and ECL. These results reveal important commonalities and differences of distinct pathway lesions, and demonstrate a pathway-specific induction of denervation-induced homeostatic synaptic plasticity.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CA1 samples from lesioned and non-lesioned organotypic entorhino-hippocampal tissue cultures.

first: Amelie

last: Eichler

first: Pia

last: Kruse

first: Phyllis

last: Stöhr

first: Dimitrios

last: Kleidonas

first: Christos

last: Galanis

first: Han

last: Lu

first: Andreas

last: Vlachos


title: ATAC-Seq analysis of the chromatin accessibilities in C9HRE ALS/FTD patient cells

summary: ATAC-seq was utilized to profile the chromatin status in ALS/FTD patient cells carrying the hexanuleotide repeat expansion in C9orf72.

overall_design: ATAC-seq was performed on control and C9orf72 HRE B lymphocytes and iPSCs-derived motor neurons.

first: Honghe


title: Chromatin accessibilities in C9HRE ALS/FTD patient cells

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: RNA-Seq based gene expression profiling of naïve B-cells from CTCFfl/+;CD19-Creki/+ mice.

summary: The goal of this study was to establish the gene expression profile of naïve B-cells from CTCFfl/+;CD19-Creki/+ mice, using RNA-seq technology.

overall_design: Naïve B cells, isolated from the spleen of CTCFfl/+;CD19-Creki/+ mice, were used to extract poly A RNA with the aim of characterizing their gene expression profiles, using RNA-Seq technology.


title: The regulative role of DAXX in the gene expression in C9HRE ALS/FTD patient motor neurons

summary: To investigate the role of DAXX in the C9HRE-associated gene dysregulation inALS/FTD patient-derived motor neurons.

overall_design: Two pairs of C9HRE ALS/FTD patient iPSCs and their isogenic controls, in which the C9Orf72 HRE mutation was removed, were differentiated into motor neurons. RNA-seq was employed to profile the global gene expression in the isogenic iMNs, C9HRE iMNs, and the C9HRE iMNs treated with shRNA targeting DAXX.


title: BdLT-Seq as a barcode decay-based method to unravel lineage-linked transcriptome plasticity

first: Maximiliano

last: PORTAL


title: DNA methylation changes from primary cultures through senescence-bypass in Syrian hamster fetal cells initially exposed to benzo[a]pyrene

city: Ottawa

first: Matthew

last: Meier

state: Ontario

address: 251 Sir Frederick Banting Driveway

department: Health Canada

institute: Government of Canada

type: Methylation profiling by high throughput sequencing

summary:
This SuperSeries is composed of the SubSeries listed below.

Purpose: Historically, the Syrian Hamster Embryo - cell transformation assay (SHE-CTA) has provided a method to predict the carcinogenicity of test chemicals. Note that the cells harvested for the SHE-CTA are from fetuses at gestation day 13.5 and not from embryos as historically recorded.  This method has been criticized for insufficient mechanistic understanding and subjective assessment of colonies morphological transformation. A more objective method is needed that additionally provides mode of action information. A possible mode of action of carcinogens includes disruption of DNA methylation in gene regulatory regions and consequent changes in gene expression.  Such genetic loci could provide biomarkers to assist in predicting the carcinogenicity of both genotoxic and non-genotoxic chemicals acting through DNA methylation disruption.

sample_organism:
Homo sapiens
Mesocricetus auratus

platform_organism:
Homo sapiens
Mesocricetus auratus

overall_design:
Refer to individual Series

This series of experiments evaluates the use of reduced representation bisulfite sequencing and RNA-seq to discover biomarkers to predict carcinogenicity.     Syrian Hamster fetal cells (SHF) were obtained using previously developed and validated methods  [OECD, 2015. Guidance document on the in vitro Syrian hamster embryo (SHE) cell transformation assay. Series on Testing & Assessment No. 214, 1-24].     The protocol described by Pickles et. al (2016) was adapted to 1) treat SHF cells with (B[a]P) or vehicle control (DMSO), 2) collect morphologically transformed (MTc) and normal (Nc) colonies, and 3) to expand colony-derived cultures to senescence (SEN) and beyond (SENbp).


title: Molecular characterization of the intact muscle spindle using a multi-omics approach

first: Bavat

last: Bornstein

address: pob 26

summary: The proprioceptive system is essential for the control of coordinated movement, posture and skeletal integrity. The sense of proprioception is produced in the brain using peripheral sensory input from receptors such as the muscle spindle, which detects changes in the length of skeletal muscles. Despite its importance, the molecular composition of the muscle spindle is largely unknown. In this study, we generated comprehensive transcriptomic and proteomic datasets of the entire muscle spindle. We then associated differentially expressed genes with the various tissues composing the spindle using bioinformatic analysis. Immunostaining verified these predictions, thus establishing new markers for the different spindle tissues. Utilizing these markers, we identified the differentiation stages the spindle capsule cells undergo during development. Together, these findings provide comprehensive molecular characterization of the intact spindle as well as new tools to study its development and function in health and disease.

overall_design: For RNA analysis, muscle spindles and adjacent extrafusal muscle fibers were collected. Muscle spindles and extrafusal fibers, adjacent to the spindle, were isolated form the deep masseter muscle. The muscle samples were used as a reference tissue by which to identify spindle-specific genes and to eliminate possible contamination with extrafusal fibers. To recognize the spindles inside the masseter belly, we marked them genetically by crossing Piezo2GFP-IRES-Cre deleter mice with a tdTomato reporter line.

first: Lia

last: Heinemann-Yerushalmi

first: Sharon

last: Krief

first: Bareket

last: Dassa

first: Dena

last: Leshkowitz

first: Guy

last: Bewick

first: Robert

middle: W

last: Banks

first: Elazar

last: Zelzer


title: Finding genetic loci that are sensitive to demethylation to assist in the development of DNA methylation-based in vitro assay for the identification of carcinogens

summary: Purpose: To  use reduced representation bisulfite sequencing and RNA-seq to  identify  demethylated promoters of genes that were derepressed following treatment with demethylating agent, 5-Aza-2'-deoxycytidine (5aCdR, CAS# 2353-33-5).

overall_design: Primary Syrian hamster fetal cells were exposed to benzo[a]pyrene for 7 days and developed morphologically transformed (MT) colonies, from which one colony was picked and reseeded and maintained in culture until it bypassed senescence. For this experiment, these immortalized cells were treated with vehicle alone or with one of two concentrations of 5aCdR (4 technical replicates for each group). Genome wide DNA methylation and mRNA expression were measured using reduced representation bisulfite sequencing (RRBS) and RNA sequencing (RNA-seq) respectively. Genes differentially induced by 5aCdR treatment whose promoters concurrently exhibited differential demethylation were considered epigenetically labile loci for further study.   All cells were confirmed male using a rapid XY qPCR assay.

first: Cathy

last: Cummings-Lorbetskie

first: Andrea

last: Rowan-Carroll

first: Daniel

last: Desaulniers


title: Low affinity CTCF binding drives transcriptional regulation whereas high affinity binding encompasses architectural functions.

first: Manuel

last: Gomez

summary:
This SuperSeries is composed of the SubSeries listed below.

CTCF is a DNA-binding protein which plays critical roles in chromatin structure organization and transcriptional regulation; however, little is known about the functional determinants of different CTCF binding sites (CBS). Using a conditional mouse model, we have identified one set of CBSs that are lost upon CTCF depletion (lost CBSs) and another set that persists (retained CBSs). Retained CBSs are more similar to the consensus CTCF binding sequence and usually span tandem CTCF peaks. Lost CBSs are enriched at enhancers and promoters and associate with active chromatin marks and higher transcriptional activity. In contrast, retained CBSs are enriched at TAD and loop boundaries. Integration of ChIP-seq and RNA-seq data has revealed that retained CBSs are located at the boundaries between distinct chromatin states, acting as chromatin barriers. Our results provide evidence that transient, lost CBSs are involved in transcriptional regulation, whereas retained CBSs are critical for establishing higher-order chromatin architecture.

overall_design:
Refer to individual Series

Naive B cells, isolated from the spleen of CTCFfl/fl;CD19-Cre ki/+ or CTCFfl/+;CD19-Cre ki/+ mice, were used to extract polyA RNA and genomic DNA with the aim of studying the effect of CTCF depletion on gene expression profiles, using RNA-Seq, and CTCF binding site occupancy, using ChIP-Seq


title: Tbx5 regulatory element controls atrial function and heart size

city: Amsterdam

first: Vincent

middle: M.

last: Christoffels

state: N/A (NA)

address: Meibergdreef 15  K2-159

department: Medical Biology

country: Netherlands

institute: Amsterdam UMC


title: Tbx5 regulatory element controls atrial function and heart size [ATAC-Seq]

summary: Cardiomyocyte nucleus ATAC-seq of atria of Tbx5(Reint) and control adult mice (n=4)

overall_design: ATAC-seq of Tbx5(REint)atria

first: Fernanda

last: Bosada

first: Karel

last: van Duijvenboden


title: Tbx5 regulatory element controls atrial function and heart size II

summary: Left atrial RNA-seq of Tbx5(REint), Tbx5(REdown), Tbx5(Reint);Prrx1(RE) and control adult mice

overall_design: Whole tissue RNA-seq of adult left atria


title: Transcriptomic Characterization of HPV+ and HPV- Head and Neck Cancer Patient-Derived Xenografts

city: San Francisco

first: Rex

middle: Hayden

last: Lee

state: CA

address: 1450 Third Street

department: Otolaryngology

institute: UCSF

summary: RNA-Seq was used to characterize a large cohort of HNSCC patient-derived xenografts (PDXs) at the transcriptional level.

overall_design: Total RNA was extracted from lysates of whole PDX tumors using the Qiagen RNeasy kit according to the manufacturer’s instructions. The total RNA was enriched for polyA transcripts and sequenced at the QB3 Genomics center at the University of California, Berkeley, using the NovaSeq 6000 platform.  After initial sequencing, the unaligned reads were derived from a combination of sources including human, mouse, viral, and ostensibly other sequences. Reads were classified to a probable source prior to mapping to the human reference and expression counting. Adapters were trimmed and reads failing QC removed using fastp (v0.20.0).  Xenome (v1.0.0), a k-mer index based pseudoaligner, classified reads as "human," "mouse," "both," "neither," or "ambiguous". Reads identified as uniquely human were advanced to the human expression pipeline. rRNA matches were removed by aligning against a ribosomal reference with bwa. The remaining reads  were mapped against an Ensembl GRCh38.90 reference, and expression counts were estimated using RSEM (v1.3.1). Library sizes were adjusted using the weighted trimmed mean of M-values method from the Bioconductor edgeR package and data were transformed to log counts per million for most analyses.  Genes with median count less than 10 were excluded. We performed a special processing of the data to identify subtypes so that our data was on the same scale as data from the Cancer Genome Atlas (TCGA). Here we adjusted the library size using the upper quartile method from the edgeR package.

middle: H

first: Aaron

last: Hechmer

first: Ritu

last: Roy

first: Adam

last: Olshen

middle: E

last: Johnson

first: Jennifer

middle: R

last: Grandis


title: RNA m6A methylome in mouse oocytes and preimplantation embryos

city: Ann Arbor

first: Kin Fai

last: Au

state: MI

address: 100 Washtenaw Avenue

department: Department of Computational Medicine and Bioinformatics

institute: University of Michigan

summary: N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNA, plays diverse regulatory roles in many biological processes and is involved in a variety of physiological behaviors. Here we present the m6A methylome maps of mouse oocytes and preimplantation embryos by a low-input approach picoMeRIP-seq. We revealed that m6A was largely and dynamically deposited on maternal and zygotic RNAs during maternal-to-zygotic transition (MZT), especially on those encoding the factors essential for cell fate determination. m6A frequently marked the maternally-derived RNAs to be degraded and the genes that were activated during zygotic genome activation. Moreover, the RNAs derived from retrotransposons, such as MTA and MERVL, were heavily occupied by m6A. Collectively, our results provide a foundation for future studies exploring the regulatory roles of m6A in mammalian early embryo development.

overall_design: Using picoMeRIP-seq we developed, the transcriptome-wide m6A maps were generated at six developmental stages including oocytes at germinal vesicle (GV) and metaphase II (MII) stages, and embryos at zygote, 2-cell, 8-cell and blastocyst stages, with two biological replicates for each stage.

first: Yunhao

last: Wang

first: Yanjiao

last: Li

first: Arne

last: Klungland

first: John

last: Dahl

first: Kin

middle: F


title: Extensive sex differences in depression-linked variants functionally assayed in mouse brain [P0&P10]

city: St. Louis

first: Joseph

last: Dougherty

state: MO

address: 660 S Euclid Ave, Campus Box 8232

department: Genetics

institute: Washington University in St. Louis

summary: Here, we selected >1000 variants from over 30 depression-associated loci using brain epigenomic data, and functionally assayed them using in vivo functional assays in the mouse brain to examine sex-by-genotype interactions. We identify extensive sex-by-allele effects in mature hippocampus, suggesting genetic risk and thus disease mechanisms may be distinct between the sexes. Unbiased informatics approaches indicated a role for nuclear hormone receptors, which was supported by .  Further, comparative analysis of allelic function in the neonatal mouse brain, during a key between developmental neonates during the masculinizing testosterone surge, and in the adult hippocampus—a region of interest in depression pathology—but not at 10 days old, a older hormonally quiescent developmental stage juveniles. Our study provides novel insights into depression genetics as influenced by age, biological sex, and cell type, and provides a framework for in vivo parallel assays at a scale not previously shown possible to functionally define interactions between sex and disease variation.

sample_organism:
Mus musculus
Adeno-associated virus 9

platform_organism:
Mus musculus
Adeno-associated virus 9

overall_design:
AAV9 MPRA library was delivered into the mouse brain in utero (embryonic day 15). Mice were screened at birth for reporter-driven red fluorescence (visible through the skull by eye by green light illumination). Brains were collected WITHOUT cerebellum for RNA extraction at postnatal day 0 (P0) (n=9 male, n=6 female) or P10 (n=7 male, 6 female).

Additional grant information: 571009 - Joseph Dougherty - Simons Foundation

first: Bernard

last: Mulvey

middle: D

first: Din

last: Selmanovic


title: Extensive sex differences in depression-linked variants functionally assayed in mouse brain [Hippocampus]

overall_design:
n=5 replicates each of AAV9-transduced adult mouse brain tissues: male total hippocampus, female total hippocampus, male Vglut1+ translating-ribosome affinity purification immunoprecipitated RNA fraction, female Vglut1+  translating-ribosome affinity purification immunoprecipitated RNA fraction.

Additional grant information: 571009 - Joseph Dougherty - Simons Foundation


title: NR2F2 melanoma cellular models

first: Igor

last: Dolgalev

address: 550 1st Ave


title: Tbx5 regulatory element controls atrial function and heart size [RNA-Seq]

first: Vincent

last: Christoffels

summary: Right and left ventricles  RNA-seq of Tbx5(REint)-/- and control E14.5 mice (n=5)

overall_design: Whole tissue RNA-seq of E14.5 ventricles

first: Fernanda

last: Bosada


title: Transcriptional analysis on the impact of our mRNA adjuvant system on monocyte derived dendritic cells

status: Public on Feb 09 2023

city: Jette

first: Kirsten

last: De Ridder

state: Brussel

address: Laarbeeklaan 103

department: Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences

country: Belgium

institute: Vrije Universiteit Brussel

summary: Targeted gene expression on human monocyte derived dendritic cells to investigate the effect of our adjuvant system on the maturation and activation using the nCounter Human PanCancer Immune Profiling Panel by Nanostring.

overall_design: Monocyte derived dendritic cells were electroporated with mRNA adjuvant system. After 24 hours the cells were prepared for RNA isolation (3 donors). As a control eGFP mRNA  was included.

first: Wout

last: De Mey

first: Hanne

last: Locy


title: The aryl hydrocarbon receptor regulates lipid mediator production in alveolar macrophages [II]

city: Neuherberg

first: Caspar

last: Ohnmacht

address: Ingolstaedter Landstrasse 1

department: Center of Allergy and Environment

institute: Helmholtz Center Munich


title: The aryl hydrocarbon receptor regulates lipid mediator production in alveolar macrophages [I]

summary:
To investigate the function of the AhR in lipid mediator production in macrophages we differentiated BMDMs from AhR-KO and wildtype mice.
We then performed gene expression profiling analysis using data obtained from RNA-seq of macrophages with and without LPS-stimulation.

overall_design: Comparative gene expression profiling analysis of RNAseq data for AhR-KO macrophages and wildtype macrophages.

first: Ann-Marie

last: Maier


overall_design: ATACseq data for AhR-KO macrophages and wildtype macrophages.

first: Anela

last: Arifovic


title: BANCseq for determination of genome-wide apparent transcription factor binding affinities [ATAC-Seq]

city: NIJMEGEN

first: Hannah

last: Neikes

state: Gelderland

address: Geert Grooteplein Zuid 28

institute: Radboud University

summary: In vivo transcription factor binding is regulated by DNA sequence and chromatin features. However, the impact of chromatin context on genome-wide transcription factor binding affinities have not yet been characterized. Here we report the establishment of a quantitative protein-DNA binding assay to determine genome-wide binding affinities to native chromatinized DNA. We use this method to quantify apparent genome-wide binding affinities of both pioneering and non-pioneering transcription factors to uncover the regulatory role of chromatin on transcription factor binding. This revealed that DNA accessibility is the main determinant of transcription factor binding, which restricts nanomolar binding of non-pioneering factors to promoters. DNA binding motifs only appear to be important for very high-affinity binding, but are not essential for nanomolar binding. We uncover numerous biological processes enriched at specific binding affinities, suggesting they are regulated at defined transcription factor concentrations. Importantly, our method adds a new quantitative layer of transcription factor biology, enabling stratification of genomic targets based on transcription factor expression and prediction of transcription factor binding sites under non-physiological conditions, such as disease associated elevated expression of (onco)genes.

overall_design: ATAC sequencing in triplicate in R1 mESC and neural progenitor cells (NPCs).

first: Katarzina

last: Kliza

first: Cathrin

last: Grawe

first: Roelof

last: Wester

first: Pascal

last: Jansen

first: Lieke

last: Lamers

first: Marijke

middle: P

last: Baltissen

first: Simon

last: van Heeringen

first: Colin

last: Logie

first: Sarah

last: Teichmann

first: Rik

middle: G

last: Lindeboom

first: Michiel

last: Vermeulen


title: BANCseq for determination of genome-wide apparent transcription factor binding affinities

summary:
This SuperSeries is composed of the SubSeries listed below.

In vivo transcription factor binding is regulated by DNA sequence and chromatin features. However, the impact of chromatin context on genome-wide transcription factor binding affinities have not yet been characterized. Here we report the establishment of a quantitative protein-DNA binding assay to determine genome-wide binding affinities to native chromatinized DNA. We use this method to quantify apparent genome-wide binding affinities of both pioneering and non-pioneering transcription factors to uncover the regulatory role of chromatin on transcription factor binding. This revealed that DNA accessibility is the main determinant of transcription factor binding, which restricts nanomolar binding of non-pioneering factors to promoters. DNA binding motifs only appear to be important for very high-affinity binding, but are not essential for nanomolar binding. We uncover numerous biological processes enriched at specific binding affinities, suggesting they are regulated at defined transcription factor concentrations. Importantly, our method adds a new quantitative layer of transcription factor biology, enabling stratification of genomic targets based on transcription factor expression and prediction of transcription factor binding sites under non-physiological conditions, such as disease associated elevated expression of (onco)genes.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Improved bacterial single-cell RNA-seq through automated MATQ-seq [bulk RNA-seq]

city: Wurzburg

first: Regan

last: Hayward

address: Josef-Schneider-Straße 2 / D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: In this work, we have improved our previously published bacterial single-cell RNA-sequencing protocol (MATQ-seq), providing enhancements that achieve a higher cell throughput while also including integration of automation. We selected a more efficient reverse transcriptase which led to a lower drop-out rate and higher workflow robustness, and we also successfully implemented a Cas9-based ribosomal RNA depletion protocol into the MATQ-seq workflow. Applying this improved protocol on a large set of single Salmonella cells sampled over growth revealed improved gene coverage and a higher gene detection limit, allowing us to reveal the expression of small regulatory RNAs such as GcvB or CsrB at a single-cell level. In addition, we were able to confirm previously described phenotypic heterogeneity in Salmonella in regards to expression of pathogenicity-associated genes.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: Bulk RNA-seq matching four growth phases used in parallel with single cell sequencing from Salmonella enterica serovar Typhimurium.

first: Christina

last: Homberger

first: Lars

last: Barquist

first: Jörg

last: Vogel


title: Improved bacterial single-cell RNA-seq through automated MATQ-seq [scRNA-seq]

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: Bacterial scRNA-seq using MATQseq of four growth phases of Salmonella enterica serovar Typhimurium.


title: Improved bacterial single-cell RNA-seq through automated MATQ-seq

summary: This SuperSeries is composed of the SubSeries listed below.


title: Effective extraction of polyribosomes from primary astrocytes exposes strategies of gene expression regulation [Heavy_complex_RNAseq]

city: Tel Aviv

first: Shir

last: Mandelboum

address: chaim levanon

department: Sagol School of Neuroscience

institute: Tel Aviv University

summary:
We optimized a polysome profiling protocol considering the unique morphology and ribosomal distribution of astrocytes.
This ‘astrocyte-optimized’ protocol was then used to study primary astrocytes treated with proinflammatory cytokines,
enabeling Riboseq analysis for measurment of the transcriptome, translatome, and translation efficiency with a coverage of over 12,000 genes.
We found several regulation strategies of gene expression in astrocytes executed as part of an extensive inflamatory response.
Enhanced translation efficiency was one of these strategies, used specifically for genes related to oxidative phosphorylation, and ribosomal proteins.

overall_design:
Three biological repetitions containing qual number of primary astrocytes were lysed using the ‘standard’ or ‘optimized’ extraction method
followed by pelleting the heavy complexes from each lysate through 1M sucrose cushion. Equal emount of mRNA from each pellet was sequenced by RNA-seq,
representing the population of transcripts that was in association with heavy  complexes (RNPs) whithin the cells.

first: Melisa

last: Herrero

first: Andrea

last: Atzmon

first: Orna

last: Elroy-Stein


title: TREM2+ and interstitial macrophages orchestrate airway inflammation in SARS-CoV-2 infection in rhesus macaques

city: Atlanta

first: Gregory

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: The COVID-19 pandemic remains a global health crisis, yet, the immunopathological mechanisms driving the development of severe disease remain poorly defined. Here, we utilize a rhesus macaque (RM) model of SARS-CoV-2 infection to delineate perturbations in the innate immune system during acute infection using an integrated systems analysis. We found that SARS-CoV-2 initiated a rapid infiltration (two days post infection) of plasmacytoid dendritic cells into the lower airway, commensurate with IFNA production, natural killer cell activation, and induction of interferon-stimulated genes. At this early interval, we also observed a significant increase of blood CD14-CD16+ monocytes. To dissect the contribution of lung myeloid subsets to airway inflammation, we generated a novel compendium of RM-specific lung macrophage gene expression using a combination of sc-RNA-Seq data and bulk RNA-Seq of purified populations under steady state conditions. Using these tools, we generated a longitudinal sc-RNA-seq dataset of airway cells in SARS-CoV-2-infected RMs. We identified that SARS-CoV-2 infection elicited a rapid recruitment of two subsets of macrophages into the airway: a C206+MRC1- population resembling murine interstitial macrophages, and a TREM2+ population consistent with CCR2+ infiltrating monocytes, into the alveolar space.  These subsets were the predominant source of inflammatory cytokines, accounting for ~75% of IL6 and TNF production, and >90% of IL10 production, whereas the contribution of CD206+MRC+ alveolar macrophages was significantly lower. Treatment of SARS-CoV-2 infected RMs with baricitinib (Olumiant®), a novel JAK1/2 inhibitor that recently received Emergency Use Authorization for the treatment of hospitalized COVID-19 patients, was remarkably effective in eliminating the influx of infiltrating, non-alveolar macrophages in the alveolar space, with a concomitant reduction of inflammatory cytokines. This study has delineated the major subsets of lung macrophages driving inflammatory and anti-inflammatory cytokine production within the alveolar space during SARS-CoV-2 infection.

overall_design: Eight rhesus macaques (Macaca mulatta) were infected with SARS-CoV-2. Four RMs were administered 4 mg Baricitinib starting at day 2 post-infection (DPI) for 8-9 consecutive days. RNA-Seq profiling of cells isolated from PBMC and BAL (bronchoalveolar lavages) prior to SARS-CoV-2 inoculation (-5 DPI; Baseline); after virus inoculation, PBMCs were isolated 1,2,4,6,7,8 DPI, and BALs were sampled 2,4,7 DPI; PBMC and BAL were samples at Necropsy 10-11 DPI.

first: Amit

last: Upadhyay

first: Timothy

middle: N

last: Hoang

first: Maria

last: Pino

first: Arun

last: Boddapati

first: Elise

last: Viox

first: Michelle

middle: Y

last: Lee

first: Jacqueline

last: Corry

first: Zachary

last: Strongin

first: David

last: Cowan

first: Elizabeth

last: Beagle

first: Tristan

last: Horton

first: Sydney

last: Hamilton

first: Hadj

last: Aoued

first: Justin

middle: L

last: Harper

first: Kevin

last: Nguyen

first: Kathryn

last: Pellegrini

first: Anne

last: Piantadosi

first: Rebecca

last: Levit

first: Rama

last: Amara

last: Barratt-Boyes

first: Susan

last: Ribeiro

first: Rafick

last: Sekaly

first: Thomas

last: Vanderford

first: Raymond

last: Schinazi

first: Mirko

last: Paiardini

first: Steven

last: Bosinger


title: The genetic background shapes the susceptibility to mitochondrial dysfunction and NASH progression

city: Lausanne

first: Giorgia

last: Benegiamo

state: Vaud

address: AI 1351 (Bâtiment AI), Station 15

country: Switzerland

institute: EPFL

summary: Non-alcoholic steatohepatitis (NASH) is a global health concern without treatment. The challenge in finding effective therapies is due to the lack of good mouse models and the complexity of the disease, characterized by gene–environment interactions. We tested the susceptibility of seven mouse strains to develop NASH. The severity of the clinical phenotypes observed varied widely across strains. PWK/PhJ mice were the most prone to develop hepatic inflammation and the only strain to progress to NASH with extensive fibrosis, while CAST/EiJ mice were completely resistant. Levels of mitochondrial transcripts and proteins as well as mitochondrial function were robustly reduced specifically in the liver of PWK/PhJ mice, suggesting a central role of mitochondrial dysfunction in NASH progression. Importantly, the NASH gene expression profile of PWK/PhJ mice had the highest overlap with the human NASH signature. Our study exposes the limitations of using a single mouse genetic background in metabolic studies and describes a novel NASH mouse model with features of the human NASH.

overall_design: RNA sequencing of male livers of 6-8 domesticated (C57BL/6J, DBA/2J, A/J, 129S1/SvlmJ) or wild-derived (CAST/EiJ, PWK/PhJ, WSB/EiJ) inbred mouse strains housed at Thermoneutrality (30°C) and fed with Western Diet or Chow Diet.

first: Giacomo

last: von Alvensleben

first: Sandra

last: Rodríguez-López

first: Ludger

middle: J

last: Goeminne

first: Alexis

middle: M

last: Bachmann

first: Jean-David

last: Morel

first: Ellen

last: Broeckx

first: Jing

last: Ma

first: Vinicius

last: Carreira

first: Sameh

middle: A

last: Youssef

first: Nabil

last: Azhar

first: Dermot

last: Reilly

first: Katharine

last: D’Aquino

first: Shannon

last: Mullican

first: Maroun

last: Bou-Sleiman

first: Johan

last: Auwerx


title: Effective extraction of polyribosomes from primary astrocytes exposes strategies of gene expression regulation [RNAseq_Riboseq]

first: Shir

last: Mandelboum

summary:
We optimized a standard polysome profiling protocol considering the unique morphology and ribosomal distribution of astrocytes.
This ‘astrocyte-optimized’ protocol was then used to study primary astrocytes treated with proinflammatory cytokines,
enabeling Ribo-seq analysis for measurment of the transcriptome, translatome, and translation efficiency with a coverage of over 12,000 genes.
We found several regulation strategies of gene expression executed as part of an extensive inflamatory response.
Enhanced translation efficiency was one of these strategies, used specifically for genes related to oxidative phosphorylation, and ribosomal proteins.

overall_design:
Three biological repetitions of primary astrocytes were treated or not with 20ng/ml TNFα and 0.25ng/ml IL-1β  for 24 hr or 48 hr.
A portion of each sample was treated with RNase-I to generate ribosome footprints used for Ribo-seq analysis, while an undigested portion was used for RNA-seq.

first: Melisa

last: Herrero Boco

first: Andrea

last: Atzmon

first: Orna

last: Elroy-Stein


title: WT and NLRP6 KO CD103+ and CD103- B cell expression

status: Public on Feb 08 2023

city: Cardiff

first: James

middle: Alexander

last: Pearson

address: Tenovus Building, School of Medicine

institute: Cardiff University

summary:
CD103+ and CD103- B cells from Wildtype Non-obese diabetic mice and NLRP6-deficient (ko) NOD mice were investigated for their gene expression
All B cells were isolated from the spleen of female non-diabetic mice aged 12-16 weeks and gated on Live, single TCRbeta-CD11c-CD11b-CD19+

overall_design: 2 WT CD103+ B cells, 2 WT CD103- B cells, 2 NLRP6 KO CD103+ B cells and 2 NLRP6 CD103- B cells were analysed


title: PI3Kβ controls immune evasion in PTEN-deficient breast tumors

city: Cambridge

first: Michelle

last: Ramseier

state: MA

address: 400 Main St

department: Harvard-MIT Health Sciences and Technology

institute: Massachusetts Institute of Technology

summary: We used scRNA-seq to characterize the anti-tumorigenic or pro-tumorigenic immune infiltrate into the tumor microenvironment of TNBC genetically engineered mouse models with PI3Ka versus PI3Kb isoform knockouts.

overall_design: Mouse tumors were dissociated, and live immune cells and live T cells were sorted to load onto Seq-Well arrays for scRNA-seq library generation.

first: Sanjay

last: Prakadan

first: Alex

middle: K

last: Shalek


title: BANCseq for determination of genome-wide apparent transcription factor binding affinities [CUT&Run]

first: Hannah

last: Neikes

overall_design: Quantification of genome wide apparent transcription factor Binding Affinities in Native Chromatin by BANC-seq was performed in human cell line MCF-7, R1 mouse embryonic stem cells (mESC), R1 neural progenitor cells (NPC), and mESC hybrid (F121, Castaneus and 129/Sv) for transcription factors YY1, FOXA1, SP1 and cMYC-MAX complex.

first: Katarzina

middle: W

last: Kliza

first: Cathrin

last: Grawe

first: Roelof

last: Wester

first: Pascal

last: Jansen

first: Lieke

last: Lamers

first: Marijke

last: Baltissen

first: Simon

last: van Heeringen

first: Colin

last: Logie

first: Sarah

last: Teichmann

first: Rik

last: Lindeboom

first: Michiel

last: Vermeulen


title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion

city: Haining

first: Heng-Jia

last: Liu

state: Zhejiang

address: 718 East Haizhou Road

department: International Campus

institute: Zhejiang University - University of Edinburgh


title: Intestinal Neuropod GUCY2C regulates visceral pain

city: Philadelphia

first: Scott

last: Waldman

state: PA

address: 1020 Locust St

department: Pharmacology and Experimental Therapeutics

institute: Thomas Jefferson University

summary: Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons, in mice and humans. GUCY2CHigh neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemically-induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.

overall_design: This data presents and compares transcriptomic profiles of GUCY2C High small intestinal "neuropod cells" to GUCY2C medium small intestinal cells.  RNAsequencing was performed on small intestines of 5, 10-week-old litter-matched Gucy2c-GFP mice that were FACS sorted into GUCY2CHigh and GUCY2CMed groups at 20,000 cells per group. mRNA was extracted using Qiagen RNeasy Micro Plus kit. Library preparation, sequencing, alignment, and read counting was performed by the Novogene Corporation (Tianjin, China). Libraries were prepared using NEBNext Ultra™ II RNA Library Prep Kit (New England Biosciences (NEB), Ipswich, MA, E7760). 50 sequencer according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on the same machine and paired-end reads were generated. STAR software was then used to align and map clean reads to the mouse reference genome (mm9).  Subsequent RNAsequencing analysis was performed in R (R Foundation for Statistical Computing, Vienna, Austria). For analysis, only genes with counts per million (cpm) >0.5 in at least 3 of the samples were included (15,689 genes). Limma (v3.40.6) and edgeR (v3.26.8) packages were used to determine differential gene expression (adj P value <0.01).

first: Joshua

last: Barton


title: Setd2 inactivation sensitizes lung adenocarcinoma to inhibitors of oxidative respiration and mTORC1 signaling [K RNAseq]

first: Amy

middle: C

last: Gladstein

state: Pennsylvania

address: 421 Curie Blvd Rm 729

department: Cancer Biology

institute: University of Pennsylvania

summary: RNA-sequencing analysis of murine K lung adenocarcinoma tumors in the presence and absence of Setd2. Tumors were harvested from K-Ctrl and K-Setd2KO mice. Tumors were then subject to RNA-Seq to identify gene expression differences following loss of Setd2 expression.

overall_design: 4 K-Ctrl and 4 K-Setd2KO  tumors were harvested from mice and then flash frozen in liquid nitrogen. The tumors were then subject to paired-end sequencing.

first: David

last: Walter

first: Katherine

last: Doerig

first: Ramakrishnan

last: Natesan

first: Saravana

last: Baskaran

first: A

last: Gudiel

first: Keren

last: Adler

first: Jonuelle

middle: O

last: Acosta

first: Douglas

last: Wallace

first: Irfan

last: Asangani

last: Feldser


title: Forced enhancer-promoter rewiring to alter gene expression in animal models

first: Gerd

last: Blobel

address: 3615 Civic Center Blvd., Abramson Research Center 316H

department: Hematology

institute: Children's Hospital of Philadelphia

summary: Transcriptional enhancers can be in physical proximity with their target genes via chromatin looping. The enhancer at the beta-globin locus (LCR) contacts the fetal (HBG) and adult (HBB) type beta-globin genes during corresponding developmental stages. We previously demonstrated that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the beta-globin locus as a model we provide proof-of-concept at the organismal level that forced enhancer re-wiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPC) from mice bearing human beta-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene regulatory elements may be used to alter gene expression to treat disease.

overall_design: RNA-Seq analysis of in vitro differentiated erythroid cells at day 13 of culture following lentiviral transduction of CD34+ primary human cells with either GFP control or ZF-Ldb1 constructs

last: Peslak


title: Network-based assessment of HDAC6 activity is highly predictive of pre-clinical and clinical responses to the HDAC6 inhibitor ricolinostat [array]

city: Memphis

first: Qingfei

last: Pan

state: TN

address: 262 Danny Thomas Place

department: Department of Computational Biology

institute: St. Jude Children's Research Hospital

summary: Despite the anticancer activity of pan-histone deacetylase (HDAC) inhibitors, their clinical use has been limited due to toxicity. However, the development of more specific inhibitors that selectively inhibit individual HDACs is emerging as a novel and well-tolerated alternative. Here, we present the results of the first clinical trial evaluating the activity of ricolinostat (the leading HDAC6 inhibitor) in breast cancer (BC) patients. We have developed a computational network-based algorithm to evaluate the activity of the HDAC6 protein, based on the enrichment of its transcriptional targets in differentially expressed genes (HDAC6 score). Through preclinical in vitro and in vivo studies, we confirmed that the HDAC6 score can stratify the sensitivity of BC cells to ricolinostat treatment and may thus have value as a predictive biomarker. Moreover, analysis of ~3,000 primary human breast cancers showed that ~30% of them present high HDAC6 scores. Based on these results, we designed a phase Ib clinical trial to evaluate the activity of ricolinostat plus nab-paclitaxel in metastatic BC patients. Study results showed that the two agents can be safely combined, that clinical activity is identified specifically in patients with HR+/HER2- disease, and that the HDAC6 score was predictive of response. Expansion of our analysis to other tumor types identified multiple cohorts enriched in high HDAC6 score samples. These results suggest that the HDAC6 score may provide an effective predictive biomarker of ricolinostat sensitivity in multiple human cancers.

overall_design: Gene expression profiles of 1) 3 breast cancer cell lines treated by ricolinostat for 6 hours 12 hours and 24 hours and by vehicle; and 2) MMTV-Neu mouse model treated by ricolinostat for 4 weeks and by vehicle.

first: Tizita

last: Zeleke

first: Kevin

last: Kalinsky

first: Jose

last: Silva

first: Jiyang

last: Yu


title: Characterizing transcriptional consequences of SETD2 inhibitor treatment in Karpas-422 cells

first: Brian

last: Liau

address: 12 Oxford St.

department: Department of Chemistry and Chemical Biology

institute: Harvard University

summary: We characterized the influence of SETD2 inhibition on gene expression in Karpas-422.

overall_design: For RNA-seq preparation, the Karpas-422 cells were subjected to vehicle (DMSO 0.1% v/v) or SETD2-IN-1 (5 µM) treatment for 10 days. Upon completion of the treatments, RNA was isolated and sequenced to assess changes in transcription.

middle: B

first: Hui

middle: S

last: Kwok

first: Allyson

last: Freedy

first: Allison

last: Siegenfeld


title: Setd2 inactivation sensitizes lung adenocarcinoma to inhibitors of oxidative respiration and mTORC1 signaling [KPY RNAseq]

first: Amy

last: Gladstein

summary: RNA-sequencing analysis of murine KPY lung adenocarcinoma tumors in the presence and absence of Setd2. Tumors were harvested from KPY-Ctrl and KPY-SetdKO mice and were then subject to RNA-Seq to identify gene expression differences following loss of Setd2 expression.

overall_design: 6 KPY-Ctrl and 8 KPY-Setd2 tumors were harvested from mice and digested into single cell suspensions,  sorted for YFP+ cells, and then flash frozen in liquid nitrogen. The tumors were then subject to single-end sequencing.

first: David

last: Walter

first: Katherine

middle: R

last: Doerig

first: Ramakrishnan

last: Natesan

last: Baskaran

last: Gudiel


title: Setd2 inactivation sensitizes lung adenocarcinoma to inhibitors of oxidative respiration and mTORC1 signaling


title: Characterizing transcriptional consequences of the CXCdel mutation in K562

summary: We characterized the effects that the CXCdel mutation had on gene expression in K562.

overall_design: RNA was isolated and sequenced to assess transcriptional differences between wild type and CXCdel knock-in K562 cells.


title: Unraveling the Gene Regulatory Networks of the Global Regulators VeA and LaeA in Aspergillus nidulans

city: Madison

first: Heungyun

middle: Harrison

last: Moon

state: WI

address: 1550 Linden drive

department: Bacteriology

institute: University of Wisconsin-Madison

overall_design: Refer to individual Series


title: Unraveling the Gene Regulatory Networks of the Global Regulators VeA and LaeA in Aspergillus nidulans [ChIP-seq]

summary: In the filamentous fungus Aspergillus nidulans, the velvet family protein VeA and the global regulator of secondary metabolism LaeA govern fungal development and secondary metabolism mostly by acting as the VelB/VeA/LaeA heterotrimeric complex. While functions of these highly conserved controllers have been well studied, the genome-wide regulatory networks   governing cellular and chemical development remain to be uncovered.  Here, by integrating transcriptomic analyses, protein-DNA interactions, and the known A. nidulans gene/protein interaction data, we have unraveled the gene regulatory networks governed by VeA and LaeA. Within the networks, VeA and LaeA directly control the expression of numerous genes involved in asexual/sexual development and primary/secondary metabolism in A. nidulans. Totals of 3,190 and 1,834 potential direct target genes of VeA and LaeA were identified, respectively, including several important developmental and metabolic regulators such as flbA·B·C, velB·C, areA, mpkB, and hogA. Moreover, by analyzing over 8,800 ChIP-seq peaks, we have revealed the predicted common consensus sequences 5’-TGATTGGCTG-3’ and 5’-TCACGTGAC-3’ that VeA and LaeA might bind to interchangeably. These findings further expand the biochemical and genomic studies of the VelB/VeA/LaeA complex functionality in gene regulation. In summary, this study unveils genes that are under the regulation of VeA and LaeA, proposes the VeA- and LaeA-mediated gene regulatory networks, and demonstrates their genome-wide developmental and metabolic regulations in A. nidulans. This entry is for the ChIP-seq data.

overall_design: Genome binding/occupancy profiling analysis by high throughput sequencing for wild-type, VeA::FLAG, and LaeA::FLAG strains.

first: Mi-Kyung

last: Lee

first: Ilhan

last: Bok

first: Jin

first: Nancy

last: Keller

first: Jae-Hyuk


title: Unraveling the Gene Regulatory Networks of the Global Regulators VeA and LaeA in Aspergillus nidulans [RNA-seq]

summary: In the filamentous fungus Aspergillus nidulans, the velvet family protein VeA and the global regulator of secondary metabolism LaeA govern fungal development and secondary metabolism mostly by acting as the VelB/VeA/LaeA heterotrimeric complex. While functions of these highly conserved controllers have been well studied, the genome-wide regulatory networks   governing cellular and chemical development remain to be uncovered.  Here, by integrating transcriptomic analyses, protein-DNA interactions, and the known A. nidulans gene/protein interaction data, we have unraveled the gene regulatory networks governed by VeA and LaeA. Within the networks, VeA and LaeA directly control the expression of numerous genes involved in asexual/sexual development and primary/secondary metabolism in A. nidulans. Totals of 3,190 and 1,834 potential direct target genes of VeA and LaeA were identified, respectively, including several important developmental and metabolic regulators such as flbA·B·C, velB·C, areA, mpkB, and hogA. Moreover, by analyzing over 8,800 ChIP-seq peaks, we have revealed the predicted common consensus sequences 5’-TGATTGGCTG-3’ and 5’-TCACGTGAC-3’ that VeA and LaeA might bind to interchangeably. These findings further expand the biochemical and genomic studies of the VelB/VeA/LaeA complex functionality in gene regulation. In summary, this study unveils genes that are under the regulation of VeA and LaeA, proposes the VeA- and LaeA-mediated gene regulatory networks, and demonstrates their genome-wide developmental and metabolic regulations in A. nidulans. This entry is for the RNA-seq data.

overall_design: Comparative gene expression profiling analysis by high throughput sequencing for wild-type, veA-deletion, and laeA-deletion strains.


title: Regulatory dissection of the severe COVID-19 risk locus introgressed by Neanderthals.

first: Terence

middle: D

last: Capellini

address: 11 Divinity Avenue

department: Human Evolutionary Biology

institute: Harvard Univserity

summary: Individuals infected with the SARS-CoV-2 virus present with a wide variety of phenotypes ranging from asymptomatic to severe and even lethal outcomes. Past research has revealed a genetic haplotype on chromosome 3 that entered the human population via introgression from Neanderthals as the strongest genetic risk factor for the severe COVID-19 phenotype. However, the specific variants along this introgressed haplotype that contribute to this risk and the biological mechanisms that are involved remain unclear. Here, we assess the variants present on the risk haplotype for their likelihood of driving the severe COVID-19 phenotype. We do this by first exploring their impact on the regulation of genes involved in COVID-19 infection using a variety of population genetics and functional genomics tools. We then perform an locus-specific massively parallel reporter assay to individually assess the regulatory potential of each allele on the haplotype in a multipotent immune-related cell line. We ultimately reduce the set of over 600 linked genetic variants to identify 4 introgressed alleles that are strong functional candidates for driving the association between this locus and the severe COVID-19. These variants likely drive the locus’ impact on severity by putatively modulating the regulation of two critical chemokine receptor genes: CCR1 and CCR5. These alleles are ideal targets for future functional investigations into the interaction between host genomics and COVID-19 outcomes.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: MPRA testing the regulatory potential of 613 variants along the introgressed risk haplotype for severe COVID-19 on chromosome 3 in K562 cells. 22 postive and 22 negative controls were also included constituting the most and least acvtive sequences in a past K562 MPRA (GEO: GSE169200). MPRA design closely followed Tewhey et al. 2016. 4 Replicates were performed.

first: Evelyn

last: Jagoda

first: Davide

last: Marnetto

first: Francesco

last: Montinaro

first: Daniel

last: Richard

first: Luca

last: Pagani

first: Gayani

last: Senevirathne

first: Victoria

last: Gonzalez

first: Kaushal

last: Baid

first: Darryl

last: Falzarano

first: Emmanuelle

middle: V

last: LeBlanc

first: Che

last: Colpitts

first: Arinjay

last: Banerjee


title: Nanowell-based single-cell combinatorial indexed ATAC-seq

city: Portland

first: Andrew

last: Adey

state: OR

address: 3181 S.W. Sam Jackson Park Rd, RHH, L103

department: MMG

institute: Oregon Health & Science University

summary: Here we present advancements in single-cell combinatorial indexed ATAC-seq (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>105) at low cost. Our optimized techniques also achieve a high fraction of reads that fall within called peaks (>80%) and low cell doublet rates. We also demonstrate an alternative workflow that achieves high cell coverage while retaining exceptional enrichment for open chromatin regions, enabling the assessment of >70,000 unique accessible loci per cell.

overall_design: Single-cell combinatorial indexed ATAC-seq using nanowell-based compartmentalization for the second tier of cell indexing (PCR)

first: Brendan

last: O'Connell


title: mTORC1 upregulates B7-H3/CD276 to inhibit antitumor T cells and drive tumor immune evasion [CITE-Seq]

first: Heng-Jia

last: Liu

summary: Immune checkpoints are often expressed on tumor cells; it remains a prevailing need to identify the mechanisms underlying their regulation and therapeutic benefit. The immune checkpoint molecule B7-H3 is upregulated in many human tumors, including those with high mechanistic/mammalian target of rapamycin complex (mTORC1) activity. However, its role in tumor immunity and the impact of B7-H3-targeted therapy on the tumor immune microenvironment are largely uncharacterized. Here, we used a syngeneic murine model of tuberous sclerosis complex (TSC), a model of mTORC1 hyperactivity, to assess the mechanisms by which B7-H3 remodels the tumor microenvironment. We performed gene and protein expression profiling analysis using data generated from CITE-seq of infiltrating CD3+ T cells isolated by FACS from subcutaneous B7-H3 knockdown and control TSC2-deficient 105K tumors.

overall_design: To sort intratumoral live CD3+ T cells, 80 control and 80 B7-H3 shRNA (1) tumors were used. Minced tumor fragments from every 300-400 mm2 of tumor were added to 3 mL of protease solution [5 mM CaCl2, 10 mg/ml Bacillus Licheniformis protease (Sigma Cat#P5380) and 125 U/mL DNase I recombinant (Roche Cat# 4716728001) in PBS and incubated with gentle rotation at 4°C for 10 min. After incubation, the solution was transferred to a Miltenyi C-tube (on ice) and the Miltenyi gentleMACS brain_03 program was run three times in a cold room (4°C). The incubation was repeated with gentle rotation at 4°C for 10 min. Single-cell dissociation was confirmed by microscopic examination. 3 mL 0.25% Trypsin-EDTA was added to the cell suspension and incubated at RT for 1 min. This was followed by the addition of 3 mL ice-cold PBS with 10% FBS. Cells were passed through a 70 µM strainer and the filter rinsed with 3 mL ice-cold PBS with 10% FBS. Tumor-infiltrating immune cells were separated from other cells by centrifugation with the deceleration brake set at 1 at 1,260 g at 4oC for 25 min in a Ficoll gradient (GE Healthcare, Cat#17-1440-03). Cells were stained with Mouse TrueStain Plus FcX (Biolegend, #156604) at 4°C for 10 min followed by staining with an anti-CD3 antibody (Biolegend, Cat#100204) for 30 min at 4°C. Tumors from each group were pooled together, then 1 x 105 live CD3+ T were sorted by flow cytometry and stained with Mouse TotalSeq-A CITE-seq antibodies (Biolegend, # 99833) at 4 °C for 30 min, before being washed in excess PBS with 2% FBS and 2.5 mM EDTA and resuspended in PBS with 0.04% BSA at 1500 cells/ml. 12000-15000 cells were loaded in the 10X Genomics Chromium in duplicate lanes.

first: Heng

last: Du

first: Damir

last: Khabibullin

first: Mahsa

last: Zarei

first: Kevin

last: Wei

first: Gordon

last: Freeman

first: David

last: Kwiatkowski

first: Elizabeth

middle: P

last: Henske


title: Effects of high cholesterol diet on intimal small RNA expression profile utilizing two different RNA-seq methods [Intima_small_RNA]

city: Riverside

first: Changcheng

last: Zhou

state: California

address: 900 University Avenue

department: Biomedical Sciences

institute: The University of California Riverside

type: Non-coding RNA profiling by high throughput sequencing

summary:
Purpose: To reveal the small RNA profiles in the intima using traditional and PANDORA-seq methods.
Methods: 3-week-old LDLR-/- male mice were fed a high cholesterol or low cholesterol diet for 9 weeks. Intimal RNA was extracted by injecting Trizol into the aorta and collecting the flow through for further processing.
Results: PANDORA-seq revealed an abundant amount of tsRNA and rsRNA that were undetected by traditional-seq. Importantly, PANDORA-seq revealed that many differentially regulated sncRNAs also undetected with traditional-seq.

overall_design: Intimal small RNA were sequenced from LDLR-/- male mice after 9 weeks of low cholesterol or high cholesterol feeding.

first: Rebecca

last: Hernandez

first: Junchao

last: Shi

first: Tong


title: Effects of high cholesterol diet on intimal transcriptome [Intima_mRNA]

summary:
Purpose: To investigate the effects of high cholesterol diet on the intimal transcriptome related to atherosclerosis.
Methods: 3-week-old LDLR-/- male mice were fed a high cholesterol or low cholesterol diet for 9 weeks. Intimal RNA was extracted by injecting Trizol into the aorta and collecting the flow through for further processing.
Results: Mice fed a high cholesterol revealed upregulation in atherosclerosis and immune/inflammatory related genes.

overall_design: Intimal mRNA and small RNA were sequenced from LDLR-/- male mice after 9 weeks of low cholesterol or high cholesterol feeding. This dataset includes the intimal mRNA data.


title: Transcriptome and chromatin accessibility mapping reveals a type I Interferon response triggered by Mycobacterium tuberculosis infection [ATAC-Seq]

city: Ottawa

first: Alexandre

last: Blais

state: ON

address: 451 Smyth Road

institute: University of Ottawa

summary: Currently there is limited knowledge of changes in genome-wide chromatin accessibility during Mycobacterium tuberculosis (Mtb) infection and whether host phosphatases such as PPM1A play a role in this process. Using combinatorial chromatin accessibility (ATAC-seq) and transcriptomics (RNA-seq) profiling of wild-type (WT), PPM1A knockout (△PPM1A) and PPM1A overexpressing (PPM1A+) macrophages, we demonstrate that Mtb infection induces global chromatin remodeling consistent with changes in gene expression signatures. The strongest concordant chromatin accessibility and gene expression signature triggered by Mtb infection was enriched for genes involved in the type I interferon (IFN) signaling pathways. Modulation of PPM1A expression results in altered chromatin accessibility signatures during Mtb infection that are reflected in the total number, chromosome location and directionality of change.

overall_design: ATAC-seq and RNA-seq for non-infected (NI) and Mtb infected wild-type (WT) THP-1, PPM1A knockout THP-1 (△PPM1A),  and PPM1A overexpressing THP-1 (PPM1A+) macrophages.

first: Jim

last: Sun

first: Alex

first: Katrina

last: Madden

first: Rayan

last: El Hamra

first: Stefania

last: Berton

first: Gonzalo

last: Alverez


title: Transcriptome and chromatin accessibility mapping reveals a type I Interferon response triggered by Mycobacterium tuberculosis infection [RNA-Seq]

last: Alvarez


title: Transcriptome and chromatin accessibility mapping reveals a type I Interferon response triggered by Mycobacterium tuberculosis infection

summary: This SuperSeries is composed of the SubSeries listed below.


title: Characterizing transcriptional consequences of EZH2 inhibitor withdrawal for Karpas-422 CXCdel cells

first: Brian

last: Liau

summary: We characterized the effects that the the withdrawal of the EZH2 inhibitor, GSK343,  has on Karpas-422 CXCdel cells

overall_design: For RNA-seq preparation, the Karpas-422 CXCdel cells grown continuously in GSK343 (1 µM) were subjected to drug removal (DMSO 0.1% v/v) or continued GSK343 (1µM) treatment for 72 hours. Upon completion of the treatments, RNA was isolated and sequenced to assess changes in transcription.

first: Hui

last: Kwok

first: Allyson

last: Freedy

first: Allison

last: Siegenfeld


title: The application of ChIP-seq to study the impact of the CXCdel mutation and SETD2 inhibition on H3K27me3 spreading.

summary: We conducted quantitative ChIP-seq to study the impact of the CXCdel mutation and SETD2 inhibition on H3K27me3 spreading. In addition, we performed standard ChIP-seq for EZH2 in wt and CXCdel Karpas-422 and K562 cell lines.

overall_design: Quantitative ChIP-seq was performed in duplicate for histone modifications following DMSO treatment or continued GSK343 treatment for Karpas-422 wt and CXCdel cell lines. For K562 cells, quantitative ChIP-seq was performed for wt and CXCdel cell lines. In addition histone modification changes for H3K36me3 and H3K27me3 were profiled following SETD2 inhibition in duplicate using quantitative ChIP-seq. Standard ChIP-seq was performed in singligate to explore the localization preferences of CXCdel EZH2 in Karpas-422 and K562 knock-in cell lines.


title: Global Transcriptional Response of Escherichia coli Exposed In Situ to Different Low-Dose Ionizing Radiation Sources

city: Newark

first: Mark

last: Blenner

state: DE

department: Chemical and Biomolecular Engineering

institute: University of Delaware

summary: Characterization of biological and chemical responses to ionizing radiation by various organisms is essential for potential applications in bioremediation, alternative modes of detecting nuclear material, and national security. Escherichia coli DH10β is an optimal system to study the microbial response to low-dose ionizing radiation at the transcriptional level because it is a well-characterized model bacterium and its responses to other environmental stressors, including those to higher radiation doses, have been elucidated in prior studies. In this study, RNA sequencing with downstream transcriptomic analysis (RNA-seq) was employed to characterize the global transcriptional response of stationary-phase E. coli subjected to Pu-239, H-3 (tritium), and Fe-55, at an approximate absorbed dose rate of 10 mGy day-1 for 1 day and 15 days. Differential expression analysis identified significant changes in gene expression of E. coli for both short- and long-term exposures. Radionuclide source exposure induced differential expression in E. coli of genes involved in biosynthesis pathways of nuclear envelope components, amino acids, and siderophores, transport systems such as ABC transporters and type II secretion proteins, and initiation of stress response and regulatory systems of temperature stress, the RpoS regulon, and oxidative stress. These findings provide a basic understanding of the relationship between low-dose exposure and biological effect of a model bacterium that is critical for applications in alternative nuclear material detection and bioremediation. IMPORTANCE Escherichia coli strain DH10β, a well-characterized model bacterium, was subjected to short-term (1-day) and long-term (15-day) exposures to three different in situ radiation sources comprised of radionuclides relevant to nuclear activities to induce a measurable and identifiable genetic response. We found E. coli had both common and unique responses to the three exposures studied, suggesting both dose rate- and radionuclide-specific effects. This study is the first to provide insights into the transcriptional response of a microorganism in short- and long-term exposure to continuous low-dose ionizing radiation with multiple in situ radionuclide sources and the first to examine microbial transcriptional response in stationary phase. Moreover, this work provides a basis for the development of biosensors and informing more robust dose-response relationships to support ecological risk assessment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for E. coli DH10β cells subjected to an absorbed dose of 10 mGy d-1 of in situ Pu-239, H-3, or Fe-55 radionuclide source or non-irradiated chemical equivalent control for one and 15-days at room temperature.

first: Molly

middle: E

last: Wintenberg

first: Lisa

last: Manglass

first: Nicole

last: Martinez


title: Identification of NLP2 in vivo targets using ChIP-Seq in Arabidopsis thaliana

city: Lyon

first: François

last: ROUDIER

address: 46 Allée d'Italie

department: Biologie

country: France

institute: Ecole Normale Supérieure de Lyon

summary: Chromatin immunoprecipitation was performed in nlp2-2 Arabidopsis thaliana Col-0 14-d-old seedlings complemented by a pNLP2::NLP2-GFP construct upon 30 minutes NO3- resupply after a 3-day NO3- starvation.

overall_design: Genome-wide profiling of NLP2 binding regions by ChIP-Seq, 2 biological replicates

last: Roudier

first: Anne

last: Krapp


title: Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

city: Leuven

first: Frederic

last: Lluis

state: Vlaams-Brabant

address: Herestraat 49

department: Development and Regeneration

institute: Stem Cell Institute Leuven

summary: Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

overall_design: RNA-seq of mESCs on day 0, 1, 2, 4, 8 of Tcf7l1 overexpression. In addition, RNA-seq was performed on mESCs after induction of EpiLCs differentiation for 3 days.

first: Paraskevi

last: Athanasouli


title: Birth and delineation of the intestinal stem cell niche

city: Boston

first: Ramesh

last: Shivdasani

address: 450 Brookline Ave. Dana 720D

department: Medical Oncology

institute: Dana Farber Cancer Institute

summary: Intestinal stem cells are sequestered into crypts generated during postnatal development in mice. During this timeframe, we investigated the transition of the surrounding mesenchymal niche. Using single cell RNA sequencing, we generated transcriptomic libraries from a combination of whole tissue and transgenically labeled fractions, including GFP+ fractions from PdgfraH2BEGFP and GFP-tDt+ fractions from PdgfraH2BEGFP;Myh11CreERT2;Rosa26tDt+ mice, of the intestinal mesenchyme at varying postnatal and adult stages. We found distinct molecular signatures of these cell types, focusing our analysis on the temporal changes and known epithelial trophic factors.

overall_design: Examination of mRNA profiles of intestinal mesenchymal cell types.

first: Neil

last: McCarthy

first: Guodong

last: Tie

first: Shariq

last: Madha

first: Adrianna

last: Maglieri

first: Judith

last: Kraiczy


title: Excitotoxic glutamate levels drive spinal cord ependymal stem cell proliferation and fate specification through CP-AMPAR signaling

city: Toronto

first: Michael

last: Fehlings

address: 60 Leonard Avenue, 7KD-430

department: Genetics and Development

institute: University Health Network

summary: The adult spinal cord contains a population of ependymal-derived neural stem/progenitor cells (epNSPCs) that are normally quiescent, but are activated to proliferate, differentiate, and migrate after spinal cord injury. The mechanisms that regulate their response to injury cues, however, remain unknown. Here, we demonstrate that excitotoxic levels of glutamate promote the proliferation and astrocytic fate specification of adult spinal cord epNSPCs through CP-AMPAR signaling.

overall_design: Bulk RNA-sequencing on rat epNSPCs in the presence and absence of excitotoxic levels of glutamate in vitro.

first: Laureen

last: Hachem

first: James

last: Hong

first: Alexander

last: Velumian

first: Andrea

last: Mothe

first: Charles

middle: H

last: Tator

middle: G