title: HDLM2_IBR_1_S4

value: pleural effusion

value: Hodgkin lymphoma

value: 48 hours


title: HDLM2_BV_2_S2

status: Public on Jun 14 2022

type: SRA

city: Syracuse

first: Frank

middle: A

last: Middleton

state: NY

address: 505 Irving Avenue

department: Neuroscience and Physiology

country: USA

institute: SUNY Upstate Medical Univ

library_source: transcriptomic

data_processing:
Stranded single-end 75bp libraries were run on the NextSeq 500 instrument and converted to FASTQ files using bcl2fastq v1.8.4 software (Illumina)
Partek Flow v9.0
5 bases were trimmed from the 5' end of each read and the 10 base sequence AAAAAAAAAA trimmed from the 3' end of each read using cutAdapt v1.12
miRNAs were identified using Bowtie1.0 alignment with an index built from hg38 miRbase22 mature sequences and normalized using median ratio (DESeq2) method
mRNAs were identified using Bowtie2 v2.2.5 alignment with an index built from hg38 RefSeq v93 sequences and normalized using median ratio (DESeq2) method
Assembly: hg38
Supplementary files format and content: DESeq2 normalized counts (miRNA)
Supplementary files format and content: DESeq2 normalized counts (mRNA)

molecule: total RNA

organism: Homo sapiens

source_name: HDLM2 cells, BV treated, 48hrs

growth_protocol: Cells cultured at 37C with 5% C02 in RPMI 1640 + 2mM Glutamine + 10 - 20% Fetal Bovine Serum

extract_protocol:
Cells were lysed in QIAzol lysis reagent (Qiagen). Following aqueous/organic phase separation, RNA was purified with the RNeasy Mini Kit (Qiagen)
For ribosomal depletion, 500ng total RNA was used as input to the NEBNext rRNA Depletion Kit v2 - Human/Mouse/Rat (New England Biolabs). The depleted RNA was used as input to the SEQuoia Complete Stranded RNA Library  Prep Kit (BioRad).

treatment_protocol: Drug treatments performed for 48 hours using empirically determined doses at or near the MIC 50 for each drug and cell line,  diluted in RPMI

tag: tissue

tag: cell line

tag: cell type

tag: time

tag: treatment

value: Brentuximab vedotin (BV) 25ng/ul


title: MNXGFP_1

city: Gothenburg

first: Lars

last: Palmqvist

address: Sahlgrenska University Hospital

department: Institute of Biomedicine

country: Sweden

institute: University of Gothenburg

data_processing:
Rna sequecning was done uwing DNBSEQ with PE100, 40M clean reads paired end
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to whole genome using STAR (version 2.9.0a) with the following parameters: outSAMtype BAM SortedByCoordinate
Quantification of counts for each gene aligned to exons was carried out using HTSeq-count (Python package HTSeq, python v 3.8.1) software using the following parameters -f bam -r pos -s no -m union with the reference annotation file. The mouse reference genome and annotation files were obtained from the Ensembl database
Differential gene expression was performed using the R package Deseq2 (version "1.31.14").  Genes with log2 (foldchange) 1 and false discovery rate (FDR) adjusted p-value < 0.05 were considered differentially expressed genes
Assembly: GRCm38
Supplementary files format and content: Raw count files obtained from HTSeq

organism: Mus musculus

source_name: Bone marrow from mice after transplantation with fetal liver cells

growth_protocol: Fetal liver hematopietic cells were obtained at embryonic days 14.5 (E14.5) and maintained in liquid culture (Dulbecco's modified Eagle's medium supplemented with 18% fetal bovine serum, 10 ng/mL human interleukin [IL]-6, 6 ng/mL murine IL-3, and 100 ng/mL murine stem cell factor). All culture media and growth factors were obtained from StemCell Technologies Inc..

extract_protocol:
Total RNA was isolated using the RNeasy Plus Mini kit (Qiagen)
mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 100) bases reads were generated in the way of sequenced by synthesis.

treatment_protocol: For treatment with Sinefungin, cells were kept in daily fresh medium with 5μM Sinefungin (in PBS) for one week after retroviral tranfection

tag: strain

value: C57BL/6 transplanted to NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice

tag: age

value: Embryonic day 14.5

tag: genotype

value: MNX1 overexpression

value: Fetal liver hematopietic cells E 14.5

value: Non-Treated


title: HDLM2_BV_1_S1


title: MNXGFP_3


title: THP-1 Cells treated with KIN1148 2.5 micromoles/liter Replicate 1

type: RNA

city: Seattle

first: Richard

last: Green

state: WA

address: 750 Republican St, Seattle, WA 98109

department: Immunology

institute: UW

description: Gene expression in THP-1 Cells after treatment with KIN1148 2.5 micromoles/liter replicate 1

hyb_protocol: The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for hybridization and array washing. Two hundred fifty ng of each RNA sample was hybridized to Mouse Whole Genome Gene Expression Microarray (G4122F) slides.

scan_protocol: Dry slides were scanned on an Agilent Technologies Scanner (Model G2505C) using the XDR setting.

data_processing: Raw images were analyzed using the Agilent Feature Extraction software (version 9.5.3.1) and the GE1-v5_95_Feb07 extraction protocol. All arrays were required to pass Agilent QC flags. Extracted raw data were background corrected using the norm-exp method and quantile normalized using Agi4x44PreProcess package.

label_protocol: The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for the Cy3-cDNA probe preparation.

extract_protocol: Total RNA purified using RNeasy kit (Qiagen) was submitted to Labcorp Seattle (previously Covance) for microarray analysis using Agilent SurePrint G3 Human Genome Microarrays (version 2).

treatment_protocol: Differentiated THP-1 cells were treated with small molecule compounds (KIN1000 and KIN1148 at 0.625, 2.5 or 10 micromoles/liter) or 25 IU/mL IFN Beta diluted in cRPMI supplemented with 0.5% (v/v) DMSO, or treated with cRPMI supplemented with 0.5% (v/v) DMSO alone (DMSO). Control cells infected with 25 HAU/mL SeV were maintained in cRPMI supplemented with 0.5% (v/v) DMSO after removal of the virus inoculum.

value: THP-1 Cells


title: IT S2_replicate 1

city: VR

first: Alessandra

last: Amato

address: strada le grazie 15

country: Italy

institute: University of Verona

hyb_protocol: Hybridization was carried out in an Agilent custom microarray (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL26142) https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126052containing 44K probes, corresponding to about 26k annotated genes plus EST and transcripts reported in the literature as candidate genes for important traits and QTLs available at NCBI databases (www.ncbi.nlm.nih.gov/gene/).

scan_protocol: Scanning and Feature Extraction was performed by using an Agilent Scanner following the settings and parameters indicated in the instruction manual

data_processing: A data-matrix was prepared selecting from each single sub-array outcome file the gProcessedSignalvalues, which are the raw fluorescence intensities of each probe.

organism: Vitis vinifera

source_name: Italia; seeds; post-bloom phase (BBCH 65-68; E-L 19-25); replicate 1

label_protocol: The cDNA synthesis, labeling hybridization, and washing were performed according to the Agilent Microarray-Based Gene Expression Analysis Guide (6.9.1)

growth_protocol: Seeded (Red Globe, Italia) and seedless (Autumn Royal, Thompson Seedless) table grapevine varieties were selected from a grape collection grown in the experimental field of the Research Centre Viticulture and Enology (CREA-VE) in Turi (BA) - South of Italy, under conventional vine management and without hormone treatments in the 2013-2014 seasons

extract_protocol: Total RNA was extracted from 0.1 g of ovules or seeds collected at each experimental stage with Total RNA Isolation Mini Kit (Agilent Technologies). RNA integrity was assessed by automated gel electrophoresis on 2100 Bioanalyzer (Agilent Technologies).

treatment_protocol: Total transcriptome assays were performed on ovules and developing seeds derived from two seeded (Italia and Red globe) and two seedless (Thompson Seedless and Autumn Royal) table grape varieties. Four developing stages were considered: pre-bloom (BBCH 57-60; E-L 17), post-bloom (BBCH 65-68; E-L 19-25), pea size (BBCH 73-75; E-L 29) and veraison (BBCH 81; E-L 35) (Ref. A.A.V.V. 1997; Lorenz D.H. at al.1994)

tag: cultivar

value: Italia

value: seeds

tag: devepmental stage


title: IT S2_replicate 2

source_name: Italia; seeds; post-bloom phase (BBCH 65-68; E-L 19-25); replicate 2


title: IT S2_replicate 3

source_name: Italia; seeds; post-bloom phase (BBCH 65-68; E-L 19-25); replicate 3


title: THP-1 Cells treated with KIN1000 2.5 micromoles/liter Replicate 1

description: Gene expression in THP-1 Cells after treatment with KIN1000 2.5 micromoles/liter replicate 1


title: HDLM2_IBR_2_S5

source_name: HDLM2 cells, IBR treated, 48hrs

value: Ibrutinib (IBR) 2.5uM


title: pleomorphic adenoma, ST_7

status: Public on Jun 13 2022

city: Hwaseong

first: Heejin

last: Kim

address: Keunjaebonggil 7

department: otorhinolaryngology-head and neck surgery

country: South Korea

institute: Hallym university of medicine

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence(using fastx_trimmer), then mapped to hsa (miRBase) using Bowtie2
Read count extraction using bedtools.
Assembly: miRBase version 21
Supplementary files format and content: tab-delimited text files include read count values for each Sample

source_name: pleomorphic adenoma

extract_protocol:
RNA was harvested using Trizol reagent (Invitrogen). Exosomal RNA was isolated using TRIzol LS reagent (Ambion). Total RNA was extracted from 200㎕ serum/plasma using miRNeasy Serum/Plasma Kit (Quaigen). Total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using NEBNext Multiplex SmallRNA Library Prep Kit (New England BioLabs) standard protocols.
small RNA-seq

value: pleomorphic adenoma

tag: tissue state

value: FFPE

tag: fraction

value: Exosomal RNA


title: HDLM2_BV_3_S3


title: THP-1 Cells treated with KIN1000 0.625 micromoles/liter Replicate 1

description: Gene expression in THP-1 Cells after treatment with KIN1000 0.625 micromoles/liter replicate 1


title: THP-1 Cells treated with KIN1000 10 micromoles/liter Replicate 1

description: Gene expression in THP-1 Cells after treatment with KIN1000 10 micromoles/liter replicate 1


title: IT S1_replicate 1

source_name: Italia; seeds; pre-bloom phase (BBCH 57-60; E-L 17); replicate 1

value: pre-bloom phase (BBCH 57-60; E-L 17)


title: MNXGFP_4


title: pleomorphic adenoma, ST_19


title: THP-1 Cells treated with KIN1148 0.625 micromoles/liter Replicate 1

description: Gene expression in THP-1 Cells after treatment with KIN1148 0.625 micromoles/liter replicate 1


title: pleomorphic adenoma, ST_8


title: pleomorphic adenoma, ST_4


title: pleomorphic adenoma, ST_3


title: pleomorphic adenoma, ST_17


title: pleomorphic adenoma, ST_18


title: IT S1_replicate 2

source_name: Italia; seeds; pre-bloom phase (BBCH 57-60; E-L 17); replicate 2


title: pleomorphic adenoma, ST_16


title: pleomorphic adenoma, ST_9


title: pleomorphic adenoma, ST_15


title: THP-1 Cells treated with DMSO Replicate 1

description: Gene expression in THP-1 Cells after treatment  with DMSO replicate 1

source_name: DMSO

value: DMSO


title: pleomorphic adenoma, ST_14


title: pleomorphic adenoma, ST_12


title: pleomorphic adenoma, ST_5


title: pleomorphic adenoma, ST_11


title: IT S1_replicate 3

source_name: Italia; seeds; pre-bloom phase (BBCH 57-60; E-L 17); replicate 3


title: pleomorphic adenoma, ST_10


title: pleomorphic adenoma, ST_1


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA9


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA6


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA8


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA4


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA13


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA3


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA12


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA5


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA7


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA1


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA10


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA8


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA4


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA11


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA9


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA7


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA5


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA13


title: pleomorphic adenoma portion of carcinoma ex pleomorphic adenoma, PA2


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA3


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA12


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA2


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA11


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA6


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA1


title: carcinoma portion of carcinoma ex pleomorphic adenoma, CA10


title: SE60-2_H3K9me3

city: Chapel Hill

first: Michael

middle: Ross

last: Kelly

state: NC

address: 120 Mason Farm Road

department: Bioinformatics and Genetics

institute: University of North Carolina at Chapel Hill

description: ChIP-Seq

library_source: genomic

data_processing:
Reads are mapped to the genome using BWA
Only reads with less than 2 mismatches are kept, and PCR duplicates are removed
Fragment density is then determiend and BigWigs are generated
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs of fragment density across the genome

molecule: genomic DNA

source_name: CRISPR-dCas9-KRAB Modified OVCAR 3 Cell Line

growth_protocol: OVCAR3 cell lines were cultured in RPMI-1640 growth media (Gibco,11875093) supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Corning)

extract_protocol: ChIP-seq for H3K9me3 was performed by Active Motif using antibody antibody 39161 with spike-in Drosophila normalization

treatment_protocol: OVCAR3 cells that stably-expressed dCas9-KRAB were transfected with sgRNAs using Fugene 6 (Promega) following the manufacturer's protocol and then fixed with 11% formaldehyde following Active Motif’s Epigenetic Services ChIP Fixation Protocol

value: OVCAR 3 (CRISPR-modified)

tag: condition

value: SE60 KRAB Targeting Rep 2

tag: chip antibody

value: anti-H3K9me3 (Active Motif, 39161)


title: SCR-1_H3K9me3

description: ChIP-Seq

value: Scramble Guide (non-targeting) Control


title: Pooled_Input

description: ChIP-Seq

value: Input (combo of all)

value: none (input)


title: OVCAR3_SE60KO2

data_processing:
Alignment to GRCh38.p12 using STAR version 2.6.0.a
Counting was done on a gene level using htseq version 0.11.2
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: The processed data is the HTSEQ counts output for each sample, before normalization. These reads were input into DESEQ2 where the normalization was performed, as such there is no "normalized" count data.

source_name: CRISPR Modified OVCAR 3 Cell Line

extract_protocol:
RNA was harvested from asynchronous cells using the Zymo Quick-RNA Miniprep Kit (Cat#R1054) with on-column DNaseI treatment
RNA-seq libraries were prepared with the Illumina TruSeq mRNA Library Kit following the manufacturer's protocol

treatment_protocol: Untreated

value: Super Enhancer 60 Deletion Clone 2


title: OVCAR3_WT2

source_name: OVCAR 3 Cell Line

value: OVCAR 3

value: Wild Type


title: OVCAR3_D9_59

data_processing:
Alignment to GRCh38.p12 using STAR version 2.6.0.a
Counting was done on a gene level using htseq version 0.11.2
Normalization was done within DESEQ2 via VST; as this was a single replicate experiment this data was read out into the normalized file DESEQ_Normalized_Output_dt_nonzero_genes.tsv.
A rank change approach was used to determine DEGs, this was done within Python 3.0 to create "OVCAR3_CRISPRI_SCREEN_DESEQ2_Normalized_with_Rank_and_Rank_Change.tsv" which contains the normalized gene values, global ranks, individual ranks, and individual vs global change in rank values for every gene across all samples.
Genome_build: GRCh38.p12
Supplementary_files_format_and_content: The processed data is the HTSEQ counts output for each sample, before normalization. These reads were input into DESEQ2 where the normalization was performed by the VST transformation; the normalized summary matrix (all counts for all samples) are located in the file called DESEQ_Normalized_Output_dt_nonzero_genes.tsv

source_name: OVCAR3 Cell Line which expresses dCas9-KRAB

extract_protocol:
RNA was harvested 72hrs after transfection of the sgRNA using the Zymo Quick-RNA Miniprep Kit (Cat#R1054) with on-column DNaseI treatment
RNA-seq libraries were contructed usng the QuantSeq 3' mRNA-seq Library Prep Kit (Lexogen) following the manufacturer's protocol

treatment_protocol: OVCAR3 cells that stably-expressed dCas9-KRAB were transfected with sgRNAs using Fugene 6 (Promega) following the manufacturer's protocol

value: OVCAR3 (expressing dCas9-KRAB)

value: Experimental KD


title: OVCAR3_SE14KO2

value: Super Enhancer 14 Deletion Clone 2


title: OVCAR3_D6_96


title: OVCAR3_D8_48


title: OVCAR3_E11_12


title: OVCAR3_D7_14


title: OVCAR3_D3_44


title: OVCAR3_D5_80


title: OVCAR3_WT1


title: OVCAR3_D12_71


title: OVCAR3_D4_84


title: OVCAR3_D2_98


title: OVCAR3_C5_33


title: OVCAR3_D10_67


title: OVCAR3_C8_FOXL2NB_gene

value: Positive Control


title: OVCAR3_D11_Scramble_2

value: Negative Control


title: OVCAR3_C6_26


title: OVCAR3_C7_77


title: SCR-2_H3K9me3

description: ChIP-Seq

value: Scramble Guide (non-targeting) Control Rep 2


title: OVCAR3_SE60KO1

value: Super Enhancer 60 Deletion Clone 1


title: SE60-1_H3K9me3

description: ChIP-Seq

value: SE60 KRAB Targeting


title: OVCAR3_C4_22


title: OVCAR3_E10_20


title: OVCAR3_C3_54


title: OVCAR3_C9_2


title: OVCAR3_D1_5


title: OVCAR3_C1_89


title: OVCAR3_C2_31


title: OVCAR3_C11_16


title: OVCAR3_C10_105


title: OVCAR3_C12_51


title: OVCAR3_B9_91


title: OVCAR3_B7_99


title: OVCAR3_B8_Empty_Vector


title: OVCAR3_B4_24


title: OVCAR3_B3_78


title: OVCAR3_B1_102


title: OVCAR3_B12_95


title: OVCAR3_A7_TP53_gene


title: OVCAR3_A6_39


title: OVCAR3_A5_21


title: OVCAR3_A4_81


title: OVCAR3_B5_10


title: OVCAR3_B11_65


title: OVCAR3_A8_50


title: OVCAR3_A2_29


title: OVCAR3_A12_55


title: OVCAR3_A1_17


title: Ctr_1

value: C57BL/6 transplanted to NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice

value: Embryonic day 14.5

value: Empty vector

value: Fetal liver hematopietic cells E 14.5

value: Non-Treated


title: OVCAR3_A3_60


title: OVCAR3_H8_36


title: OVCAR3_A10_87


title: OVCAR3_A11_RNF4_Enh


title: OVCAR3_B6_41


title: OVCAR3_H7_61


title: OVCAR3_H9_25


title: OVCAR3_H4_86


title: OVCAR3_H5_88


title: OVCAR3_H2_79


title: OVCAR3_H3_43


title: OVCAR3_B2_73


title: OVCAR3_H12_49


title: OVCAR3_B10_45


title: OVCAR3_A9_6


title: OVCAR3_H1_RNF4_gene


title: OVCAR3_G7_100


title: OVCAR3_H10_DORM_1


title: OVCAR3_G8_23


title: OVCAR3_G9_13


title: OVCAR3_G6_69


title: OVCAR3_G1_82


title: OVCAR3_F9_83


title: OVCAR3_G4_92


title: OVCAR3_H11_18


title: OVCAR3_G12_104


title: OVCAR3_G2_32


title: OVCAR3_F8_9


title: OVCAR3_F7_106


title: OVCAR3_F6_56


title: OVCAR3_F4_47


title: OVCAR3_F12_94


title: OVCAR3_G3_53


title: OVCAR3_H6_63


title: OVCAR3_G11_Scramble_1


title: OVCAR3_F3_38


title: OVCAR3_G10_8


title: OVCAR3_F10_19


title: OVCAR3_F2_64


title: OVCAR3_E8_42


title: OVCAR3_F11_4


title: OVCAR3_F1_101


title: OVCAR3_E7_72


title: OVCAR3_F5_FOX_Enh


title: OVCAR3_G5_58


title: OVCAR3_E9_85


title: OVCAR3_E5_90


title: OVCAR3_E6_75


title: OVCAR3_E2_62


title: Z47

city: Linköping

first: Lech

middle: Tadeusz

last: Kaczmarczyk

address: Campus US

department: Cell Biology

institute: Linköping University

data_processing:
Basecalls performed using CASAVA version 1.8.2
RNA-seq reads were aligned to the mouse reference genome (mm10) with Bowtie2 (v2.0.2) using RSEM (v1.2.29) with default parameters
Genome_build: mm10
Supplementary_files_format_and_content: The mouse reference genome was indexed using the Ensembl annotations (v84) with rsem-prepare-reference from RSEM software. Next, rsem-calculate-expression was used to align the reads and quantify the gene abundance. The raw count from rsem-calculate-expression is used.

source_name: SST

extract_protocol:
Each frozen brain hemisphere was weighed and put into a tissue grinder with a corresponding volume of polysome buffer (50mM Tri pH = 7.5, 100mM KCl, 12mM MgCl2, 1% Nonidet P-40, 1mM DTT, 1x Protease inhibitor (SigmaFast Protease Inhibitor Tablets (S8820), Sigma-Aldrich), 100U/ml RNAse inhibitor (RNAase inhibitor (N8080119), Thermo Fisher Scientific), 100µg/mL cycloheximide (Cycloheximide (C7698), Sigma-Aldrich) to prepare a 10% brain homogenate. Homogenate was prepared with help of a motorized tissue grinder (Heidolph, 600rpm, ~30s) on ice and centrifuged (10000g, 10min, 4°C). Supernatant was transferred to a new reaction tube and used as IP input and for isolation of total RNA. For IP with magnetic beads (Dynabeads Protein G 10004D, Thermo Fisher Scientific) pre-cleared supernatant (25µl beads and 200µl supernatant, 30min, 4°C) was first incubated with anti-HA antibody 12CA5 (Roche Life Science; 200µl pre-cleared supernatant, 10µl antibody, 45min, 4°C) and this mixture then added to the magnetic beads (50µl beads, 1-2h, 4°C). Magnetic beads were washed three times with PBS (~500µl) before use and incubation steps of IP were done on a rotator. IP samples were put on a magnetic rack and the magnetic bead pellets were washed three times with high salt buffer (50mM Tris pH = 7.5, 300mM KCl, 12mM MgCl2, 1% Nonidet P-40, 1mM DTT, 100µg/ml cycloheximide; ~500µl). Cell-type-specific mRNA was eluted from the magnetic beads by RLT buffer supplemented with 2-mercaptoethanol from the RNeasy Mini Kit (Quiagen; 200µl; Thermomixer: 700rpm, 5-10min, RT) and afterwards isolated with this kit. For each supernatant two technical replicates of IP were done, and total RNA from the input supernatant (200µl) was isolated in parallel. Quality and quantity of immunoprecipitated mRNA and total RNA were verified by Qubit Fluorometer (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies).
300ng of total RNA or 150ng of immunoprecipitated mRNA was used for RNA-sequencing. Each sample was checked for amount and quality using a Nanodrop 2000 (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer. For each condition (5 Cre/Rtag lines, injected with RML or NBH, sacrificed at 10 or 18 WPI) we used four individual samples (exceptions: SST RML 10 WPI: n = 3; PV NBH 10 WPI: n = 3; Astro RML 18 WPI: n = 2). RNA was converted to cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Aplied Science). RNA-sequencing libraries were prepared using the TruSeq RNA Sample Preparation 2 Kit (Illumina). The library quality was checked using an Agilent 2100 Bioanalyzer and concentration was measured by a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and adjusted to 2nM before sequencing (single end, 50 bp) on a HiSeq 2000 Sequencer (Illumina) using TruSeq SR Cluster Kit 3-cBot-HS (Illumina) and TruSeq SBS Kit 3-HS (Illumina) according to the manufacturer’s instructions.

value: RML

tag: timepoint

value: 18 weeks

tag: tag

value: RiboTag

value: SST


title: Z46x47

value: Input


title: OVCAR3_E3_30

value: OVCAR3 (expressing dCas9-KRAB)

value: Experimental KD


title: Z37x42

value: Control


title: Z39

value: 10 weeks


title: Z38x39


title: Z44x45


title: Z38


title: Z45


title: Z36


title: Z35x36


title: Z37


title: OVCAR3_E1_PLAG1_gene

value: Positive Control


title: Z35


title: Z42


title: Z46


title: OVCAR3_E12_28


title: OVCAR3_E4_52


title: Z32x33


title: Z32


title: Z29


title: Z24


title: Z44


title: Z25


title: Z24x25


title: U96


title: U77


title: U69


title: U77x96


title: U57


title: U58


title: Z27x29


title: U54


title: U37x45


title: U68x69


title: U57x58


title: S5x7


title: U38x39


title: U12


title: U68


title: U45


title: U14


title: U38


title: U37


title: U53x54


title: U13x14


title: U53


title: U13


title: S7


title: U39


title: S5


title: U11x12


title: S49


title: U11


title: S23


title: Z33


title: S45


title: S21


title: S1S49


title: S23x30


title: S30


title: S18


title: S1S45


title: S177


title: S176x177


title: S16


title: S16x21


title: S176


title: S11x13


title: S11


title: E679

source_name: PV

value: PV


title: E680


title: E678


title: E677


title: E675


title: E674


title: S179


title: E224


title: S13


title: E211


title: E208


title: E192


title: E191


title: E190


title: Kidney sample 189

city: Columbus

first: Kellie

middle: J.

last: Archer

state: OH

address: 1841 Neil Ave

department: Division of Biostatistics

institute: Ohio State University

description: Robust multiarray average probe set expression summaries from donor kidney biopsies

hyb_protocol: Following the Affymetrix HG-133Av2 GeneChip protocol

scan_protocol: The array image was generated by the high-resolution GeneChip® Scanner 3000 by Affymetrix.

data_processing: The Bioconductor affy package was used to obtain the Robust Multiarray Average probe set expression summaries using the rma function.

label: biotin

molecule: polyA RNA

source_name: Pre-implantation donor kidney biospy

label_protocol: Briefly, total RNA was reverse-transcribed using T7-polydT primer and converted into double-stranded cDNA, with templates being used for an in vitro transcription reaction to yield biotin-labeled antisense cRNA. The labeled cRNA was chemically fragmented and made into the hybridization cocktail according to the Affymetrix GeneChip protocol, which was then hybridized to Affymetrix® HG-133Av2 GeneChip.Porcine Gene 1.0 ST Array GeneChips

extract_protocol: Total RNA from kidney donor tissue samples was extracted using a miRNeasy Mini Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The purity and integrity of the isolated RNA was assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, MA).

tag: kdpi

tag: donor age

tag: donor race

tag: donor bmi

tag: batch effect


title: D695

tag: cell type


title: Kidney sample 191

tag: gfr group

value: good


title: E223

tag: strain


title: Kidney sample 190


title: Kidney sample 183


title: Kidney sample 184


title: Kidney sample 186

value: poor


title: Kidney sample 182


title: Kidney sample 181


title: Kidney sample 192


title: Kidney sample 185


title: Kidney sample 180


title: Kidney sample 187


title: D694


title: Kidney sample 179


title: Kidney sample 178


title: Kidney sample 188


title: D693


title: D692


title: D690


title: Kidney sample 177


title: D688


title: Kidney sample 176


title: S179x18


title: Kidney sample 174


title: Kidney sample 175


title: Kidney sample 173


title: D687


title: Kidney sample 172


title: D215


title: D213


title: D214


title: D176


title: D198


title: D175


title: D203


title: D689


title: Kidney sample 168


title: LN229 S4-2

city: Dalian

first: dapeng

last: Liang

state: Liaoning

address: lvshun south road no.9

country: China

institute: Dalian Medical University

data_processing:
Clean data were obtained by trimming adapter and low quality reads from raw fastq data using fastp with --length_required 50 and --detect_adapter_for_pe options
Clean reads were aligned to the hg38 genome assembly using STAR
STAR with --quantMode option was used to generate gene-level count matrix as input for DESeq2 statistical model
Convert Ensembl IDs to Entrez IDs and discard any unmapped features. Multimapped Ensembl IDs were also discarded to enforce unique mapping. Only features with unique Ensembl ID to Entrez ID mappings were retained.
Differential expression analysis between LN229-Control and LN229-S4 cells was performed using DESeq2 package
Assembly: hg38
Supplementary files format and content: csv file include gene raw count and gene annotation

growth_protocol: LN229 cells were routinely generated by re-plating with DMEM supplied with 5% FBS.

extract_protocol:
RNA was isolated from LN229-Control and LN229-S4 using TRIzol Reagent.
The sequencing libraries were prepared using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer's instructions.

treatment_protocol: LN229 cells were  treated with DMSO(0.6 μl/ml) and S4 (60 μM).

value: glioblastoma cells

tag: tissue

value: brain


title: Kidney sample 167


title: Kidney sample 171


title: HepG2/shPJA/TGFb1

city: Houston

first: Jian

last: Chen

state: TX

address: 1515 Holcombe Boulevard - Unit 1466

institute: The University of Texas M. D. Anderson Cancer Center

data_processing:
Whole-transcriptome RNA sequencing of  HepG2-shCtrl,HepG2-shPJA1, HepG2/Ctrl and HepG2/TGFb1 cells was carried out on an Illumina HiSeq 2000 sequencer.
The raw data quality was assessed using a FastQC software.
Adaptor presence was tested using Trimmomatic.
The readings were then aligned to the NCBI mouse reference genome build 37.2 using the splice-aware aligner Tophat2 v2.0.10.
Transcript quantification, normalization and assembly were carried out with Cufflinks.
A gene model gtf file corresponding to the NCBI mouse reference genome build 37.2 was used in the quantification.
Cuffdiff2, part of the Cufflinks suite of tools, was used to identify significant differences in gene expression profiles between the MEF cells.
Supplementary_files_format_and_content: FPKM tab-delimited text file format with fold change comparison and p-value calculation

source_name: Liver cancer cells

growth_protocol: HepG2 cells were infected with Lenti-virus mediated shRNA targeting human PJA1 gene (sc-91297) or contorl shRNA (Santa Cruz Biotechnology, sc-108080). The gene knockdown was confirmed by Q-PCR. HepG2 cells were treated by TGF-beta1 at 200pM for 1 hour.

extract_protocol:
Total RNA was extracted using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: treatment

value: HepG2 cell treattd with water

tag: cell line


title: LN229 S4-1


title: LN229 Control-2


title: LN229 Control-1


title: D204


title: HepG2/shPJA1

value: HepG2 cell infected with shRNA-PJA1


title: HepG2/shCtrl

value: HepG2 cell infected with shRNA-contril


title: Kidney sample 170


title: Kidney sample 169


title: Kidney sample 164


title: Kidney sample 163


title: Kidney sample 166


title: Kidney sample 158


title: Kidney sample 160


title: HepG2/TGFb1

value: HepG2 cell treattd with TGFb1


title: Kidney sample 165


title: Kidney sample 156


title: Kidney sample 159


title: Kidney sample 161


title: sybrgold-test: unstained 4np (SGE4)

city: Oxford

first: Robert

last: Beagrie

address: Headley Way

department: Molecular Haematology Unit

country: United Kingdom

institute: MRC Weatherall Institute of Molecular Medicine

data_processing:
Library strategy: Genome Architecture Mapping
Reads were aligned to the mm9 build of the mouse genome using bowtie v2.1.
GAMtools (https://gam.tools) was used to discard poorly or non-uniquely mapped reads, remove PCR duplicates and call positive 40kb windows based on the level of read coverage. Samples with less than 15% of reads mapping to the Mouse genome were not taken forward for further analysis but can be found in a separate GEO submission under the same super-series.
Genome_build: mm9
Supplementary_files_format_and_content: GAM segregation table: Each row represents one 40kb window from the mouse genome, each column indicates one sequencing sample (containing three nuclear profiles). Values indicate whether the given window was present (1) or absent (0) from the given sample.

source_name: Mouse ES Cells

growth_protocol: Cells were grown at 37°C in a 5% CO2 incubator in Glasgow Modified Eagles Medium, supplemented with 10% fetal bovine serum, 2 ng/ml LIF and 1 mM 2-mercaptoethanol, on 0.1% gelatin-coated dishes. Cells were passaged every other day. After the last passage 24h before harvesting, ES cells were re-plated in serum-free ESGRO Complete Clonal Grade medium (Millipore Inc.)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each). Staining was performed with sterile-filtered SYBR‚Ñ¢ Gold (10000x diluted in H2O) for 10 min, followed by 2 washes with water (30 s each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Samples were then frozen and stored overnight at -20 ∞C. The next day, whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: Kidney sample 162


title: sybrgold-test: unstained 4np (SGG3)


title: Kidney sample 151


title: OVCAR3_WT3

description: RNA-seq

value: OVCAR 3

value: Wild type


title: sybrgold-test: unstained 4np (SGE3)


title: OVCAR3_SE14KO1

description: RNA-SEQ

value: OVCAR 3 (CRISPR-modified)

value: Super Enhancer 14 Deletion Clone 1 Rep 1


title: sybrgold-test: unstained 4np (SGC3)


title: Kidney sample 153


title: sybrgold-test: unstained 4np (SGB3)


title: Kidney sample 154


title: sybrgold-test: unstained 4np (SGD4)


title: sybrgold-test: unstained 4np (SGF4)


title: sybrgold-test: unstained 4np (SGF3)


title: Kidney sample 148


title: large-vs-small-kit: large-kit 4np (LKC4)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: Kidney sample 146


title: Kidney sample 157


title: sybrgold-test: unstained 4np (SGD3)


title: sybrgold-test: unstained 4np (SGA3)


title: large-vs-small-kit: large-kit 4np (LKH3)


title: large-vs-small-kit: large-kit 4np (LKA4)


title: large-vs-small-kit: large-kit 4np (LKB4)


title: Kidney sample 142


title: large-vs-small-kit: large-kit 4np (LKG3)


title: Kidney sample 149


title: large-vs-small-kit: large-kit 4np (LKE3)


title: Kidney sample 145


title: Kidney sample 141


title: Kidney sample 152


title: large-vs-small-kit: large-kit 4np (LKD3)


title: Kidney sample 147


title: Kidney sample 144


title: large-vs-small-kit: large-kit 4np (LKC3)


title: Kidney sample 150


title: Kidney sample 143


title: large-vs-small-kit: large-kit 4np (LKB3)


title: large-vs-small-kit: large-kit 4np (LKA3)


title: large-vs-small-kit: large-kit 4np (LKH2)


title: large-vs-small-kit: small-kit 4np (LKF2)


title: Kidney sample 140


title: Kidney sample 138


title: Kidney sample 137


title: Kidney sample 139


title: Kidney sample 136


title: Kidney sample 134


title: Kidney sample 135


title: Kidney sample 132


title: large-vs-small-kit: small-kit 4np (LKE2)


title: large-vs-small-kit: small-kit 4np (LKD2)


title: large-vs-small-kit: small-kit 4np (LKB2)


title: large-vs-small-kit: small-kit 4np (LKC2)


title: large-vs-small-kit: small-kit 4np (LKH1)


title: large-vs-small-kit: small-kit 4np (LKA2)


title: Kidney sample 130


title: Kidney sample 131


title: eosin-test: unstained 4np (EOC6)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: large-vs-small-kit: small-kit 4np (LKF1)


title: large-vs-small-kit: small-kit 4np (LKD1)


title: large-vs-small-kit: small-kit 4np (LKE1)


title: large-vs-small-kit: small-kit 4np (LKB1)


title: eosin-test: unstained 4np (EOB6)


title: eosin-test: unstained 4np (EOA6)


title: Kidney sample 133


title: Kidney sample 127


title: Kidney sample 126


title: Kidney sample 125


title: large-vs-small-kit: small-kit 4np (LKG1)


title: Kidney sample 129


title: Kidney sample 123


title: eosin-test: unstained 4np (EOF5)


title: eosin-test: unstained 4np (EOG5)


title: Kidney sample 124


title: Kidney sample 128


title: Kidney sample 121


title: Kidney sample 119


title: eosin-test: unstained 4np (EOD5)


title: Kidney sample 122


title: Kidney sample 117


title: Kidney sample 118


title: Kidney sample 120


title: Kidney sample 113


title: Kidney sample 114


title: eosin-test: unstained 4np (EOB5)


title: eosin-test: unstained 4np (EOA5)


title: Kidney sample 116


title: eosin-test: unstained 4np (EOG4)


title: eosin-test: unstained 4np (EOC5)


title: eosin-test: unstained 4np (EOF4)


title: eosin-test: unstained 4np (EOE4)


title: Kidney sample 112


title: eosin-test: unstained 4np (EOH5)


title: Kidney sample 111


title: eosin-test: unstained 4np (EOD4)


title: Kidney sample 107


title: Kidney sample 115


title: Kidney sample 108


title: Kidney sample 105


title: eosin-test: unstained 4np (EOC4)


title: cresyl-violet-test: cresyl-violet 4np (CVH6)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each). Staining was performed with sterile-filtered cresyl violet (1 % w/v in water) for 10 min, followed by 2 washes with water (30 s each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: eosin-test: unstained 4np (EOH4)


title: Kidney sample 106


title: cresyl-violet-test: cresyl-violet 4np (CVF6)


title: Kidney sample 109


title: cresyl-violet-test: cresyl-violet 4np (CVG6)


title: Kidney sample 110


title: cresyl-violet-test: cresyl-violet 4np (CVE6)


title: cresyl-violet-test: cresyl-violet 4np (CVD6)


title: cresyl-violet-test: cresyl-violet 4np (CVC6)


title: Kidney sample 103


title: cresyl-violet-test: cresyl-violet 4np (CVA6)


title: Kidney sample 101


title: Kidney sample 155


title: cresyl-violet-test: cresyl-violet 4np (CVH5)


title: Kidney sample 102


title: Kidney sample 100


title: Kidney sample 98


title: Kidney sample 95


title: cresyl-violet-test: cresyl-violet 4np (CVG5)


title: Kidney sample 96


title: Kidney sample 104


title: eosin-test: unstained 4np (EOE5)


title: Kidney sample 93


title: Kidney sample 99


title: Kidney sample 92


title: Kidney sample 94


title: cresyl-violet-test: cresyl-violet 4np (CVE5)


title: cresyl-violet-test: cresyl-violet 4np (CVC5)


title: cresyl-violet-test: cresyl-violet 4np (CVB6)


title: cresyl-violet-test: cresyl-violet 4np (CVF5)


title: cresyl-violet-test: cresyl-violet 4np (CVD5)


title: cresyl-violet-test: cresyl-violet 4np (CVB5)


title: Kidney sample 90


title: Kidney sample 89


title: Kidney sample 88


title: Kidney sample 91


title: cresyl-violet-test: cresyl-violet 4np (CVA5)


title: cresyl-violet-test: unstained 4np (CVF2)


title: cresyl-violet-test: unstained 4np (CVE2)


title: Kidney sample 86


title: cresyl-violet-test: unstained 4np (CVB2)


title: Kidney sample 87


title: cresyl-violet-test: unstained 4np (CVC2)


title: cresyl-violet-test: unstained 4np (CVG1)


title: cresyl-violet-test: unstained 4np (CVH1)


title: cresyl-violet-test: unstained 4np (CVF1)


title: cresyl-violet-test: unstained 4np (CVD1)


title: cresyl-violet-test: unstained 4np (CVE1)


title: Kidney sample 84


title: Kidney sample 82


title: Kidney sample 81


title: Kidney sample 80


title: wga-pcr-cycles-test: 20-pcr-cycles 4np (F43F3)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 20 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: wga-pcr-cycles-test: 20-pcr-cycles 4np (F43E3)


title: wga-pcr-cycles-test: 20-pcr-cycles 4np (F43C3)


title: wga-pcr-cycles-test: 20-pcr-cycles 4np (F43B3)


title: wga-pcr-cycles-test: 23-pcr-cycles 4np (F43F2)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 23 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: Kidney sample 78


title: wga-pcr-cycles-test: 23-pcr-cycles 4np (F43E2)


title: cresyl-violet-test: unstained 4np (CVA1)


title: wga-pcr-cycles-test: 23-pcr-cycles 4np (F43D2)


title: Kidney sample 76


title: Kidney sample 83


title: Kidney sample 77


title: Kidney sample 79


title: wga-pcr-cycles-test: 23-pcr-cycles 4np (F43C2)


title: wga-pcr-cycles-test: 23-pcr-cycles 4np (F43B2)


title: Kidney sample 75


title: Kidney sample 74


title: Kidney sample 72


title: Kidney sample 73


title: wga-pcr-cycles-test: 25-pcr-cycles 4np (F43C1)


title: Kidney sample 71


title: early-freezing-test: frozen-early 4np (F42D4)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Samples were then frozen and stored overnight at -20 ∞C. The next day, whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: Kidney sample 85


title: Kidney sample 69


title: Kidney sample 70


title: Kidney sample 68


title: early-freezing-test: frozen-early 4np (F42E4)


title: Kidney sample 65


title: Kidney sample 66


title: Kidney sample 64


title: early-freezing-test: frozen-early 4np (F42C4)


title: Kidney sample 97


title: wga-pcr-cycles-test: 25-pcr-cycles 4np (F43F1)


title: Kidney sample 63


title: Kidney sample 67


title: Kidney sample 62


title: early-freezing-test: frozen-early 4np (F42G3)


title: early-freezing-test: frozen-early 4np (F42B4)


title: early-freezing-test: frozen-early 4np (F42E3)


title: Kidney sample 61


title: early-freezing-test: frozen-early 4np (F42H3)


title: early-freezing-test: frozen-early 4np (F42F3)


title: early-freezing-test: frozen-early 4np (F42D3)


title: Kidney sample 60


title: early-freezing-test: frozen-early 4np (F42C3)


title: Kidney sample 59


title: early-freezing-test: frozen-early 4np (F42A3)


title: Kidney sample 58


title: Kidney sample 57


title: early-freezing-test: frozen-late 4np (F42E2)


title: early-freezing-test: frozen-late 4np (F42D2)


title: Kidney sample 53


title: Kidney sample 54


title: Kidney sample 51


title: early-freezing-test: frozen-late 4np (F42B2)


title: early-freezing-test: frozen-late 4np (F42C2)


title: Kidney sample 52


title: early-freezing-test: frozen-late 4np (F42H1)


title: Kidney sample 55


title: early-freezing-test: frozen-early 4np (F42B3)


title: Kidney sample 50


title: Kidney sample 56


title: Kidney sample 49


title: early-freezing-test: frozen-late 4np (F42F1)


title: early-freezing-test: frozen-late 4np (F42E1)


title: early-freezing-test: frozen-late 4np (F42D1)


title: Kidney sample 48


title: early-freezing-test: frozen-late 4np (F42A1)


title: Kidney sample 43


title: early-freezing-test: frozen-late 4np (F42B1)


title: early-freezing-test: frozen-late 4np (F42C1)


title: Kidney sample 44


title: Kidney sample 45


title: full-collection-4np: 4np (G1E12)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each). Staining was performed with cresyl violet (1 % w/v in water) for 10 min, followed by 2 washes with water (30 s each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Caps were frozen and stored for up to two weeks. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: full-collection-4np: 4np (G1G11)


title: Kidney sample 47


title: full-collection-4np: 4np (G1E11)


title: eosin-test: unstained 4np (EOB4)


title: Kidney sample 42


title: Kidney sample 46


title: Kidney sample 41


title: Kidney sample 40


title: full-collection-4np: 4np (G1B11)


title: Kidney sample 39


title: full-collection-4np: 4np (G1A11)


title: full-collection-4np: 4np (G1E10)


title: full-collection-4np: 4np (G1G10)


title: Kidney sample 35


title: full-collection-4np: 4np (G1H9)


title: full-collection-4np: 4np (G1G9)


title: full-collection-4np: 4np (G1D11)


title: full-collection-4np: 4np (G1F9)


title: Kidney sample 37


title: Kidney sample 36


title: full-collection-4np: 4np (G1E9)


title: Kidney sample 33


title: full-collection-4np: 4np (G1B9)


title: Kidney sample 34


title: Kidney sample 31


title: Kidney sample 32


title: full-collection-4np: 4np (G1E8)


title: Kidney sample 29


title: full-collection-4np: 4np (G1H7)


title: full-collection-4np: 4np (G1G7)


title: Kidney sample 25


title: Kidney sample 28


title: Kidney sample 26


title: full-collection-4np: 4np (G1E7)


title: wga-pcr-cycles-test: 25-pcr-cycles 4np (F43D1)


title: full-collection-4np: 4np (G1C9)


title: full-collection-4np: 4np (G1D7)


title: full-collection-4np: 4np (G1C7)


title: Kidney sample 24


title: full-collection-4np: 4np (G1A9)


title: Kidney sample 30


title: full-collection-4np: 4np (G1D6)


title: full-collection-4np: 4np (G1F6)


title: full-collection-4np: 4np (G1E6)


title: Kidney sample 23


title: full-collection-4np: 4np (G1H5)


title: full-collection-4np: 4np (G1G5)


title: Kidney sample 16


title: Kidney sample 17


title: Kidney sample 21


title: Kidney sample 27


title: Kidney sample 20


title: Kidney sample 19


title: Kidney sample 22


title: full-collection-4np: 4np (G1F5)


title: Kidney sample 18


title: Kidney sample 15


title: full-collection-4np: 4np (G1E5)


title: Kidney sample 14


title: full-collection-4np: 4np (G1C5)


title: full-collection-4np: 4np (G1D5)


title: full-collection-4np: 4np (G1B5)


title: full-collection-4np: 4np (G1B6)


title: full-collection-4np: 4np (G1E4)


title: Kidney sample 13


title: Kidney sample 12


title: full-collection-4np: 4np (G1F4)


title: full-collection-4np: 4np (G1D4)


title: Kidney sample 10


title: Kidney sample 9


title: Kidney sample 11


title: full-collection-4np: 4np (G1B4)


title: Kidney sample 7


title: Kidney sample 8


title: full-collection-4np: 4np (G1G3)


title: full-collection-4np: 4np (G1H3)


title: full-collection-4np: 4np (G1F3)


title: full-collection-4np: 4np (G1C3)


title: Kidney sample 6


title: Kidney sample 5


title: full-collection-4np: 4np (G1D3)


title: full-collection-4np: 4np (G1B3)


title: full-collection-4np: 4np (G1A3)


title: full-collection-4np: 4np (G1F2)


title: full-collection-4np: 4np (G1G2)


title: full-collection-4np: 4np (G1E2)


title: full-collection-4np: 4np (G1H1)


title: full-collection-4np: 4np (G1D2)


title: Kidney sample 1


title: Kidney sample 3


title: Kidney sample 2


title: Kidney sample 4


title: full-collection-4np: 4np (G1G1)


title: LMZ RA-depleted mid-gastrula, biological rep3

city: PARIS

first: Jean-Francois

last: RIOU

address: 9, quai Saint-Bernard, case 24

department: Institut de Biologie Paris-Seine

country: France

institute: CNRS/Sorbonne Universite

description: Gene expression from lateral marginal zone explants at the origin of lateral mesoderm and pronephric kidney field

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip™ Xenopus laevis Genome 2.0 Array . GeneChips  were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the ThermofisherGeneChip Scanner 3000 7G.

data_processing: CEL files were read and converted to an expression measure using RMA in R (justRMA function of Affy R package)

organism: Xenopus laevis

source_name: GFP-Cyp26-injected Xenopus embryo

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2005-2009, Affymetrix).

growth_protocol: LMZ explants are dissected at early gastrula stage from embryos previously injected at the 4-cell stage. Explants are collected 1h, 3h after dissection (culture 20°C) and overnight (culture 14)C)

extract_protocol: Quiagen RNeasy kit according to manufacturer instructions

treatment_protocol: Control RNA mix (GFP) or RA-depletion mix (GFP and cyp26) are injected equatorially into the 4 blastomeres at the 4-cell stage

tag: developmental stage

value: Mid-gastrula stage


title: LMZ RA-depleted early neurula, biological rep3

value: Early neurula stage


title: LMZ control early neurula, biological rep3

source_name: GFP-injected Xenopus embryo

value: GFP-injected


title: LMZ control mid-gastrula, biological rep3


title: LMZ RA-depleted early gastrula, biological rep3

value: Early gastrula stage


title: full-collection-4np: 4np (G1E1)


title: full-collection-4np: 4np (G1F1)


title: LMZ control early neurula, biological rep2


title: LMZ RA-depleted early neurula, biological rep2


title: LMZ control early gastrula, biological rep3


title: LMZ RA-depleted mid-gastrula, biological rep2


title: full-collection-4np: 4np (G1D1)


title: freezing-after-incubation: frozen-post-incubation 4np (F41C4)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. Samples were then frozen and stored overnight at -20 ∞C. The next day, 4.32 µl library preparation mastermix (2.88 µl library preparation buffer, 1.44 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 1.4 µl library preparation enzyme was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 87.84 µl amplification master mix (10.8 µl amplification buffer, 69.84 µl H2O, 7.2 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: full-collection-4np: 4np (G1C2)


title: full-collection-4np: 4np (G1C1)


title: freezing-after-incubation: frozen-post-incubation 4np (F41H3)


title: freezing-after-incubation: frozen-post-incubation 4np (F41B4)


title: freezing-after-incubation: frozen-post-incubation 4np (F41G3)


title: freezing-after-incubation: frozen-post-incubation 4np (F41E3)


title: freezing-after-incubation: frozen-post-incubation 4np (F41F3)


title: freezing-after-incubation: frozen-post-incubation 4np (F41B3)


title: 1 np: (f14g2)

extract_protocol:
DNA extraction and whole genome amplification were carried out using the sigma WGA4 kit, following the protocol in Beagrie et al. 2017
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: 1 np: (f14f5)


title: 1 np: (f14f6)


title: LMZ control mid-gastrula, biological rep1


title: LMZ control early neurula, biological rep1


title: LMZ RA-depleted early neurula, biological rep1


title: LMZ RA-depleted early gastrula, biological rep1


title: 1 np: (f14g5)


title: LMZ RA-depleted mid-gastrula, biological rep1


title: LMZ control mid-gastrula, biological rep2


title: LMZ control early gastrula, biological rep2


title: LMZ RA-depleted early gastrula, biological rep2


title: freezing-after-incubation: unfrozen 4np (F41D2)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.32 µl library preparation mastermix (2.88 µl library preparation buffer, 1.44 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 1.4 µl library preparation enzyme was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 87.84 µl amplification master mix (10.8 µl amplification buffer, 69.84 µl H2O, 7.2 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: freezing-after-incubation: frozen-post-incubation 4np (F41C3)


title: LMZ control early gastrula, biological rep1


title: freezing-after-incubation: unfrozen 4np (F41C2)


title: 1 np: (f14f3)


title: 1 np: (f14f1)


title: freezing-after-incubation: unfrozen 4np (F41A2)


title: 1 np: (f14f4)


title: 1 np: (f14e7)


title: freezing-after-incubation: unfrozen 4np (F41F1)


title: 1 np: (f14g1)


title: freezing-after-incubation: unfrozen 4np (F41B2)


title: 1 np: (f14f7)


title: freezing-after-incubation: unfrozen 4np (F41G1)


title: 1 np: (f14e6)


title: Kidney sample 38


title: 1 np: (f14e3)


title: 1 np: (f14e2)


title: freezing-after-incubation: unfrozen 4np (F41B1)


title: 1 np: (f14d5)


title: freezing-after-incubation: unfrozen 4np (F41D1)


title: 1 np: (f14d6)


title: 1 np: (f14e4)


title: freezing-after-incubation: frozen-post-incubation 4np (F41D4)


title: 1 np: (f14d7)


title: freezing-after-incubation: unfrozen 4np (F41C1)


title: freezing-after-incubation: frozen-post-incubation 4np (F41E4)


title: freezing-after-incubation: unfrozen 4np (F41A1)


title: 1 np: (f14d2)


title: pk-volume-titration: 2ul-mdc-pk 4np (F40F3)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (12.9 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.2 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 5.35 µl library preparation mastermix (3.57 µl library preparation buffer, 1.78 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 5.35 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 108.9 µl amplification master mix (13.4 µl amplification buffer, 86.6 µl H2O, 8.9 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: pk-volume-titration: 2ul-mdc-pk 4np (F40G3)


title: pk-volume-titration: 2ul-mdc-pk 4np (F40E3)


title: 1 np: (f14c5)


title: 1 np: (f14c6)


title: pk-volume-titration: 2ul-mdc-pk 4np (F40D3)


title: pk-volume-titration: 1ul-mdc-pk 4np (F40G2)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 5.35 µl library preparation mastermix (3.57 µl library preparation buffer, 1.78 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 5.35 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 108.9 µl amplification master mix (13.4 µl amplification buffer, 86.6 µl H2O, 8.9 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: 1 np: (f14c4)


title: 1 np: (f14c7)


title: pk-volume-titration: 2ul-mdc-pk 4np (F40B3)


title: pk-volume-titration: 1ul-mdc-pk 4np (F40F2)


title: pk-volume-titration: 2ul-mdc-pk 4np (F40A3)


title: freezing-after-incubation: unfrozen 4np (F41E1)


title: 1 np: (f14c3)


title: 1 np: (f14d3)


title: pk-volume-titration: 1ul-mdc-pk 4np (F40D2)


title: 1 np: (f14b7)


title: pk-volume-titration: 1ul-mdc-pk 4np (F40C2)


title: 1 np: (f14b3)


title: 1 np: (f14b6)


title: wga-reagent-volumes: protocol-c 4np (F37E6)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Caps were frozen and stored for up to two weeks. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 5.35 µl library preparation mastermix (3.57 µl library preparation buffer, 1.78 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 5.35 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 108.9 µl amplification master mix (13.4 µl amplification buffer, 86.6 µl H2O, 8.9 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: wga-reagent-volumes: protocol-b 4np (F37E4)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Caps were frozen and stored for up to two weeks. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: 1 np: (f14b2)


title: 1 np: (f14d1)


title: wga-reagent-volumes: protocol-a 4np (F37D2)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Caps were frozen and stored for up to two weeks. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.32 µl library preparation mastermix (2.88 µl library preparation buffer, 1.44 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 1.4 µl library preparation enzyme was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 87.84 µl amplification master mix (10.8 µl amplification buffer, 69.84 µl H2O, 7.2 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: 1 np: (f14a5)


title: pi-staining-and-freezing: unstained 4np (F38H1)


title: 1 np: (f14a4)


title: wga-reagent-volumes: protocol-b 4np (F37D4)


title: 1 np: (f14a7)


title: 1 np: (f14a3)


title: pi-staining-and-freezing: unstained 4np (F38H2)


title: 1 np: (f14c1)


title: 1 np: (f14b1)


title: pi-staining-and-freezing: unstained 4np (F38G1)


title: pi-staining-and-freezing: unstained 4np (F38F2)


title: wga-reagent-volumes: protocol-c 4np (F37D6)


title: 1 np: (f14b4)


title: 1 np: (f14d4)


title: pi-staining-and-freezing: unstained 4np (F38F1)


title: 1 np: (f13g5)


title: wga-reagent-volumes: protocol-a 4np (F37E2)


title: 1 np: (f13g4)


title: pi-staining-and-freezing: unstained 4np (F38G2)


title: 1 np: (f13f6)


title: 1 np: (f14a6)


title: pi-staining-and-freezing: unstained 4np (F38E1)


title: pk-volume-titration: 1ul-mdc-pk 4np (F40A2)


title: 1 np: (f14a2)


title: pi-staining-and-freezing: unstained 4np (F38E2)


title: 1 np: (f13f5)


title: pi-staining-and-freezing: unstained 4np (F38D3)


title: pi-staining-and-freezing: unstained 4np (F38D1)


title: 1 np: (f13f1)


title: pi-staining-and-freezing: unstained 4np (F38C2)


title: pi-staining-and-freezing: unstained 4np (F38C3)


title: pi-staining-and-freezing: unstained 4np (F38D2)


title: pi-staining-and-freezing: unstained 4np (F38C1)


title: 1 np: (f13g1)


title: pi-staining-and-freezing: unstained 4np (F38B2)


title: pi-staining-and-freezing: unstained 4np (F38E3)


title: pi-staining-and-freezing: unstained 4np (F38B1)


title: 1 np: (f13e6)


title: 1 np: (f13f3)


title: 1 np: (f13e4)


title: 1 np: (f14a1)


title: 1 np: (f13e2)


title: wga-reagent-volumes: protocol_c 4np (F37G5)


title: 1 np: (f13d3)


title: wga-reagent-volumes: protocol_c 4np (F37E5)


title: pi-staining-and-freezing: unstained 4np (F38B3)


title: 1 np: (f13d6)


title: 1 np: (f13c5)


title: 1 np: (f13c4)


title: wga-reagent-volumes: protocol_c 4np (F37C6)


title: wga-reagent-volumes: protocol_c 4np (F37B6)


title: 1 np: (f13d1)


title: 1 np: (f13d4)


title: pi-staining-and-freezing: unstained 4np (F38A2)


title: 1 np: (f13d2)


title: pi-staining-and-freezing: unstained 4np (F38A3)


title: wga-reagent-volumes: protocol_c 4np (F37H5)


title: wga-reagent-volumes: protocol_c 4np (F37D5)


title: 1 np: (f13f2)


title: 1 np: (f13f4)


title: wga-reagent-volumes: protocol_b 4np (F37D3)


title: 1 np: (f13b6)


title: 1 np: (f13b5)


title: 1 np: (f13b4)


title: wga-reagent-volumes: protocol_b 4np (F37C4)


title: wga-reagent-volumes: protocol_c 4np (F37A6)


title: wga-reagent-volumes: protocol_c 4np (F37C5)


title: 1 np: (f13c1)


title: 1 np: (f13c3)


title: wga-reagent-volumes: protocol_b 4np (F37E3)


title: wga-reagent-volumes: protocol_b 4np (F37H3)


title: 1 np: (f13c2)


title: wga-reagent-volumes: protocol_b 4np (F37B3)


title: 1 np: (f13b3)


title: wga-reagent-volumes: protocol_b 4np (F37C3)


title: wga-reagent-volumes: protocol_b 4np (F37A4)


title: 1 np: (f13c6)


title: 1 np: (f13a1)


title: 1 np: (f13a4)


title: 1 np: (f14g6)


title: wga-reagent-volumes: protocol_a 4np (F37D1)


title: wga-reagent-volumes: protocol_a 4np (F37B2)


title: 1 np: (f13a5)


title: 1 np: (f14g3)


title: 1 np: (f13b2)


title: 1 np: (f14f2)


title: wga-reagent-volumes: protocol_a 4np (F37H1)


title: wga-reagent-volumes: protocol_b 4np (F37B4)


title: 1 np: (f13b1)


title: 1 np: (f14e5)


title: 1 np: (f14g4)


title: 1 np: (f13a3)


title: 1 np: (f13a2)


title: sybrgold-test: unstained 4np (SGC4)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each). Staining was performed with sterile-filtered SYBR‚Ñ¢ Gold (10000x diluted in H2O) for 10 min, followed by 2 washes with water (30 s each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Samples were then frozen and stored overnight at -20 ∞C. The next day, whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: wga-reagent-volumes: protocol_a 4np (F37A1)


title: wga-reagent-volumes: protocol_b 4np (F37A3)


title: wga-reagent-volumes: protocol_a 4np (F37B1)


title: wga-reagent-volumes: protocol_a 4np (F37C1)


title: wga-reagent-volumes: protocol_a 4np (F37G1)


title: 1 np: (f14c2)


title: wga-reagent-volumes: protocol_a 4np (F37A2)


title: 1 np: (f13g3)


title: 1 np: (f14b5)


title: 1 np: (f13g2)


title: sybrgold-test: unstained 4np (SGB4)


title: sybrgold-test: unstained 4np (SGA4)


title: sybrgold-test: unstained water-control (SGH3)

extract_protocol:
An adhesive 8-strip LCM collection cap was left empty during laser microdissection to act as a negative control. Whole genome amplification was performed on the empty cap using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: 1 np: (f13e5)


title: 1 np: (f14e1)


title: 1 np: (f13e3)


title: sybrgold-test: stained-sybrgold 4np (SGH2)


title: 1 np: (f13e1)


title: sybrgold-test: stained-sybrgold 4np (SGG2)


title: sybrgold-test: stained-sybrgold 4np (SGE2)


title: sybrgold-test: stained-sybrgold 4np (SGF2)


title: 1 np: (f13d5)


title: sybrgold-test: stained-sybrgold 4np (SGD2)


title: 1 np: (f13a6)


title: sybrgold-test: stained-sybrgold 4np (SGH1)


title: sybrgold-test: stained-sybrgold water-control (SGA2)


title: Bacteria injection - ultrasound Sham R231

city: Niskayuna

first: John

last: Graf

state: New York

address: One Research Circle

institute: GE Global Research

data_processing:
Fastq files of the paired end reads were merged into two files for each sample, one for R1 and one for R2 using Unix commands.
Base quality control was checked using Fast QC v0.10.1 from Babraham Bioinformatics.
Sequencing reads were mapped to the annotated rat genome version, Rattus norvegicus Rnor 6.0.91, using STAR_2.5.3a aligner.
Transcript abundance estimates were generated from the sorted BAM files using RSEM.
Transcript abundance normalization was performed on all samples using the DESeq2 tool.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: rsem_genes.csv : comma-delimited text file that includes the table of RSEM output for every gene and every sample
Supplementary_files_format_and_content: rsem_isoforms.csv : comma-delimited text file that includes the table of RSEM output for every transcript and every sample
Supplementary_files_format_and_content: transcript_read_counts.csv : comma-delimited text file that includes the table of raw read counts for every transcript and every sample
Supplementary_files_format_and_content: transcript_normalized_abundance.csv : comma-delimited text file that includes the table of Median Ratio normalization (MRN) abundances for every transcript and every sample

organism: Rattus norvegicus

source_name: blood 18 hours post injection and 2 hours post ultrasound

growth_protocol: Adult male Sprague-Dawley rats (age 9-11 weeks) weighing 300-400 gm were used in this study under the approval of Institutional Animal Care and Use Committee (IACUC) at the Feinstein Institutes for Medical Research. Streptococcus Pneumoniae Serotype 19F (49619, American Type Culture Collection (ATCC)) was cultured in Brain Heart Infusion broth until it reached the log phase with an optic density of 0.5 at 600nm. It was then aliquoted in 1.8 ml cryogenic vials with 30% glycerol and stored in a -80C freezer.

extract_protocol:
Blood was collected at the terminal time point 2 hours post ultrasound treatment from cardiac puncture. RNA extraction from tissues and RNA sequencing were performed at the Feinstein Institutes for Medical Research (FIMR), Genomics Shared Resource, Manhassett, New York.
Sequencing RNA libraries were prepared using the Rat TruSeq Stranded Total RNA with Ribo-Zero Globin Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s instructions.

treatment_protocol: Lung instillation was performed on rats anesthetized with isoflurane (5% for induction and 2-3% for maintenance). A catheter was placed in the trachea below the larynx (IV Catheter 20G x 1-3/4in L, Jelco). A polyethylene tube (PE-10) was then inserted inside the catheter. Either saline or a bacterial dose of 3E5 suspended in 200 ul saline was instilled. Splenic ultrasound stimulation was delivered at 16 hours after bacteria or saline inoculation. The rats were anesthetized using isoflurane. The location of the spleen was identified using diagnostic imaging ultrasound. Animals either received 5 minutes of ultrasonic stimulus (1.1 MHz, 200mV per pulse, 150 burst cycles, 200 ms burst period) or Sham stimulus where no energy was applied. The ultrasound system consists of RF power amplifier (E&I 350L), a function generator (Agilent 33120A), a matching network (Sonic Concept), and High-Frequency Focused Ultrasound Probe/transducer (Sonic Concept H106).

value: blood

tag: injection

value: Bacteria

tag: ultrasound

value: Sham

tag: series


title: sybrgold-test: stained-sybrgold 4np (SGC2)


title: mES_Brd2_ChIA-PET_rep1

city: Beijing

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

department: School of Life Sciences

institute: Peking University

data_processing:
Library strategy: ChIA-PET
The raw sequences from the paired-end sequencing were analyzed for the presence of the PET linker barcodes and trimmed using Cutadapt with the parameters “-m 17 - forward=ACGCGATATCTTATCTGACT -a reverse=AGTCAGATAAGATATCGCGT --overlap 10” (Martin, 2011)
Specifically, we trimmed the 3’ sequence immediately after a perfect match of at least the first 10nt of the linker sequences. After removing the linkers and the 3’ sequence following the linkers, only the 5’ ends of the trimmed PETs of at least 17bp in length were retained for effective mapping to the reference genome.
Only the uniquely mapped reads with at most a single mismatch were retained, the PET sequences that had PET linkers on at least one end of the PET were used for further analysis.
The sequences of the two ends of PET sequences were separately mapped to the mm9 mouse genome using the bowtie algorithm with the option “-k 1 -m 1 -v 1”.
PET sequences with identical genomic coordinates and strand information at both ends were collapsed into a single PET sequence.
Genome_build: mm9
Supplementary_files_format_and_content: High-confidence interactions

source_name: mouse embryonic stem cells

growth_protocol: The V6.5 mouse ES (mES) cell line was cultured with irradiated mouse embryonic fibroblasts in DMEM-KO containing 15% FBS, leukaemia inhibiting factor (LIF), L-glutamine, β-mercaptoethanol, non-essential amino acids and penicillin/ streptomycin at 37 ℃ with 5% CO2, and passage twice for experiments.

extract_protocol:
We modified ChIA-PET protocol based on previously described version (Ji et al., 2016). Briefly, the nuclei were sonicated and incubated with specific antibody and it was the same as the procedure described in the ChIP-Seq, and then, after proximal ligation of ChIP-DNA fragments, tagmentation of ligated products and purification, DNA were amplificated by PCR.
ChIA-PET library was generated by Illumina HiSeq 2000.

treatment_protocol: Wild type mES cells were untreated.

value: mES

tag: genotype

value: WT

tag: antibody


title: Bacteria injection - ultrasound Sham R230


title: sybrgold-test: stained-sybrgold 4np (SGB2)


title: sybrgold-test: stained-sybrgold 4np (SGG1)


title: mES_Brd2_ChIA-PET_rep2


title: sybrgold-test: stained-sybrgold 4np (SGF1)


title: sybrgold-test: stained-sybrgold 4np (SGE1)


title: Bacteria injection - ultrasound Sham R229


title: Bacteria injection - ultrasound Stimulation R228

value: Stimulation


title: Bacteria injection - ultrasound Stimulation R227


title: sybrgold-test: stained-sybrgold 4np (SGA1)


title: sybrgold-test: stained-sybrgold 4np (SGC1)


title: Bacteria injection - ultrasound Stimulation R224


title: Bacteria injection - ultrasound Sham R232


title: Bacteria injection - ultrasound Sham R221


title: sybrgold-test: stained-sybrgold 4np (SGB1)


title: large-vs-small-kit: large-kit water-control (LKD4)


title: large-vs-small-kit: large-kit 4np (LKF3)

extract_protocol:
220 nm cryosections were collected in sucrose droplets and transferred to steel frame PEN membrane slides. Slides were washed in 1x PBS (3 times, 5 min each), then with water (3 times, 5 min each) and air dryed for 15 minutes. Nuclear profiles were laser microdissected into adhesive 8-strip LCM collection caps, with four profiles dissected into each cap. Whole genome amplification was performed using the Sigma WGA4 kit. 14.5 µl of lysis and fragmentation mastermix (13 µl H2O, 1.4 µl lysis and fragmentation buffer, 0.1 µl proteinase K) was added to each well of a 96 well plate, caps with microdissected material were used to close the wells, then the plate was inverted and centrifuged upside-down at 3000 xg for 2 min such that the fragmentation mastermix was collected in the cap. Plates were incubated upside-down for 4 h at 50 ∞C then inverted and centrifuged right-way-up at 3000 xg for 2 min to collect the extracted DNA in the bottom of the well. Samples were then heat inactivated at 99 ∞C for 4 min then cooled on ice for 2 min. 4.95 µl library preparation mastermix (3.3 µl library preparation buffer, 1.65 µl library stabilisation solution) was added to each sample, incubated at 95 ∞C for 2 min, cooled on ice for 2 min then centrifuged at 3000 xg for 2 min. 4.5 µl library preparation enzyme (diluted 3x with H2O) was added to each tube, then samples were incubated at 16 ∞C for 20 min, then 24 ∞C for 20 min, 37 ∞C for 20 min and 75 ∞C for 5 min. Finally, 85 µl amplification master mix (11 µl amplification buffer, 66.5 µl H2O, 7.5 µl WGA polymerase) was added to each tube and samples were amplified by PCR (initial denaturation 95 ∞C for 3 min, then 25 cycles of denaturation 95 ∞C for 30 s and annealing/extension 65 ∞C for 5 min).
WGA amplified DNA fragments larger than 500bp were purified using Ampure XP beads and prepared for sequencing using the Illumina Nextera kit, following the manufacturer's guidelines


title: large-vs-small-kit: small-kit water-control (LKG2)