title: spt6-1004 37C TSS-seq 2

description: Transcription Start Site Sequencing (TSS-seq) of the spt6-1004 mutant S. cerevisiae strain grown at 30ºC and shifted to 37ºC for 80 minutes.

data_processing:
Library strategy: TSS-seq
The Snakemake pipeline used to process TSS-seq libraries is available at github.com/winston-lab. Removal of adapter sequences from the 3’ end of the read and 3’ quality trimming were performed using cutadapt. The random hexamer molecular barcode on the 5’ end of the read was removed and processed using a custom Python script. Reads were aligned to the combined S. cerevisiae and S. pombe reference genomes using Tophat2 without a reference transcriptome. Uniquely mapping reads were selected using SAMtools. Reads mapping to the same location as another read with the same molecular barcode were identified as PCR duplicates and removed using a custom Python script. Coverage of the 5’-most base, corresponding to the TSS, was extracted using bedtools genomecov and normalized to the total number of reads uniquely mapping to the S. pombe genome.
Genome_build: Saccharomyces cerevisiae S288C R64-2-1, Schizosaccharomyces pombe ASM294v2
Supplementary_files_format_and_content: TSS-seq “plus” and “minus” files are bigWig files of spike-in normalized S. cerevisiae transcription start sites on the positive and negative strands, respectively. ChIP-nexus “plus” and “minus” files are bigWig files of library-size normalized S. cerevisiae coverage of the points of crosslinking on the positive and negative strand, respectively. ChIP-nexus “midpoint” and “protection” files are library-size normalized S. cerevisiae coverage of either the midpoint of the fragment predicted to be protected by the ChIP’ed factor by MACS2, or the entire fragment. NET-seq “plus” and “minus” files are bigWig files of library-size normalized S. cerevisiae coverage of the positions of transcribing RNA polymerase II on the positive and negative strands, respectively. MNase-seq “midpoint” files are bigWig files of spike-in normalized S. cerevisiae dyad coverage determined by taking the midpoint of each sequenced fragment. MNase-seq “midpoint_smoothed” files are bigWig files of the same spike-in normalized dyad coverage, smoothed with a Gaussian kernel of 20bp bandwidth. MNase-seq “wholefrag” files are bigWig files of spike-in normalized S. cerevisiae  coverage of the entire fragment protected from MNase digestion.

extract_protocol:
Cell concentration was determined using a hemocytometer. S. pombe cells (strain FWP10) were added as spike-in (10% the number of cerevisiae  cells). Total RNA was isolated as previously described (Ausubel, 1991). Poly(A)-enriched RNA was isolated from 300μg of total RNA with 300μL of Dynabeads oligo(dT)25, using the manufacturer’s instructions and eluting in water.
Prior to each step of library construction, RNA samples were heat denatured at 80ºC for two minutes and rapidly cooled on ice, followed by addition of 40 U of RNasin (Promega). Between each enzymatic reaction, samples were purified using an RNA binding column (Zymo Research). Ten to fifteen μg of poly(A) RNA was dephosphorylated with 30 units of calf intestinal phosphatase (CIP; NEB) for one hour at 37ºC. CIP was removed from the reaction by heat inactivation followed by phenol extraction, and traces of phenol were removed using the above-mentioned RNA column. The m7GpppN cap was then cleaved from the RNA with 12.5 units of CapClip (CELLSCRIPT) for one hour at 37ºC and the decapped RNA, containing a 5’ monophosphate, was ligated to 25 pmoles of DNA/RNA chimeric linker (oSDAP4) containing a randomized RNA linker sequence of six nucleotides at the 3’ end and a 5’ biotin moiety in a 10μL reaction with 20 units of T4 RNA ligase 1 (NEB) and 2mM ATP. Ligation products were column purified as before and eluted into fragmentation buffer (Ingolia, 2009) calibrated to enrich for 90-120 nucleotide oligomers. Fragmented RNA was then size selected and purified from a 10% acrylamide urea gel (Invitrogen). PNK removal of the 3’ phosphate group and 3’-end ligation fo the RNA to a random linker pool (Mayer, 2015) was done as previously described (Couvillion, 2017), except after ligation the biotinylated RNA was affinity purified with 10μL of Dynabeads M-270 streptavidin (Invitrogen) using the manufacturer’s instructions. Bead-bound RNA was eluted into 50μL of elution buffer (0.1% SD, 10mM Tris 7.5) at 90ºC for 5 minutes, and reverse transcribed with 3 pmoles of RT primer (oSMDRT2) by heating for 5 min at 65ºC, with 200 units SSIII Reverse Transcriptase (Invitrogen) at 48ºC for 45 minutes. The cDNA was gel purified as above, and PCR amplified for 10-14 cycles using previously described indexing and sequencing primers for Illumina sequencing (Couvillion, 2017).

value: polyA and 5’ capped RNA


title: NaClrep2

status: Public on Nov 01 2018

type: SRA

city: Cuernavaca

first: Joaquin

last: Sanchez

state: Morelos

address: Calle Ixtaccihuatl Esq Leñeros

department: Facultad de Medicina

country: Mexico

institute: Universidad Autónoma del Estado de Morelos

library_source: transcriptomic

data_processing:
none provided by the submitter
Genome_build: NC_011601.1
Supplementary_files_format_and_content: raw counts and RPKM

molecule: total RNA

organism: Escherichia coli

source_name: Bacteria

extract_protocol: At the end of the incubation period, cultures were transferred to ice and bacterial cells were collected at 4°C by centrifugation at 10,000 x g for a 10 min period. The collected cells were then subjected to RNA extraction.

tag: culture

value: Exposed to NaCl


title: spt6-1004 37C TSS-seq 1

city: Boston

first: Fred

last: Winston

state: MA

address: 77 Avenue Louis Pasteur, Room 239

department: Genetics

country: USA

institute: Harvard Medical School

organism: Saccharomyces cerevisiae

source_name: log phase S. cerevisiae

growth_protocol: Cells were grown in YPD at 30ºC to approximately 1x107 cells/ml (OD600=0.6), at which point an equal volume of YPD medium pre-warmed to 44ºC was added, and the cultures were shifted to 37ºC for an additional 80 minutes.

tag: strain

tag: temperature

tag: tissue

value: log phase S. cerevisiae

tag: experiment type

value: Transcription Start Site Sequencing (TSS-seq)

tag: target molecule


title: Mouse 12.1

city: Edinburgh

first: Liam

last: Morrison

state: Midlothian

address: Easter Bush

country: United Kingdom

institute: The Roslin Institute, The University of Edinburgh

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_12_1

data_processing:
Library strategy: cDNA Amplicon sequencing
Pacbio raw data was initially processed using the Pacbio SMRT analysis protocol (v2.3)
Raw data was converted into a fastq file using read_of_insert protocol with the following parameter selections: minimum 1 full pass, minimum predicted accuracy of 90%.
Based on the read length distribution and predicted gene length, a range of 1400-2000bp was used to filter the sequenced reads for downstream VSG analysis in fasta format
VSG variant distribution was determined by locally aligning the reads to TREU927 reference transcripts annotated to ‘VSGs’ using custom workflows
Genome_build: TREU927 genome (v26) on www.tritrypdb.org
Supplementary_files_format_and_content: the VSG transcript counts for the aligned reads are presented in the tab-seperated ".txt" file

organism: Trypanosoma brucei brucei

source_name: Infected mouse blood

extract_protocol:
Mice were maintained for 12 days post-infection, and groups of 5 mice were euthanased at 3, 6, 10 and 12 days post-infection. At each sampling day, RNA was extracted from 200 µl infected blood using the Qiagen RNeasy kit. Approximately 1 µg RNA was treated with DNase Turbo and cDNA was generated via reverse transcription. VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3UTR).
DNA was purified with 1x cleaned Ampure beads (Agencourt) and the quantity and quality was assessed using Nanodrop and Qubit assay. Fragment Analyser (using a high sensitivity genomic kit) was used to determine the average size of the DNA and the extent of degradation. DNA was treated with Exonuclease V11 at 37 °C for 15 minutes. The ends of the DNA were repaired as described by the Pacific Biosciences protocol. Samples were incubated for 20 minutes at 37 °C with damage repair mix supplied in the SMRTbell library kit (Pacific Biosciences). This was followed by a 5 minute incubation at 25 °C with end repair mix. DNA was cleaned using 0.5x Ampure beads and 70% ethanol washes. DNA was ligated to adapter overnight at 25 °C. Ligation was terminated by incubation at 65°C for 10 minutes followed by exonuclease treatment for 1 hour at 37°C. The SMRTbell libraries were purified with 0.5x Ampure beads. The quantity of library and therefore the recovery was determined by Qubit assay and the average fragment size determined by Fragment Analyser. SMRTbell libraries were then annealed to the sequencing primer at values predetermined by the Binding Calculator (Pacific Biosciences) and a complex made with the DNA Polymerase (P4/C2chemistry). The complex was bound to Magbeads and this was used to set up 20 SMRT cells for sequencing. Sequencing was done using 180 minute movie times.

treatment_protocol: Mice were inoculated with 1 x 10^5 Trypanosoma brucei brucei TREU927 via the intraperitoneal route.

tag: host organism

value: Mus musculus BALB/c


title: S8BR

type: RNA

city: Tokushima

first: Taigo

last: Horiguchi

address: 3-18-15, Kuramoto-Cho

country: Japan

institute: Tokushima University

description:
Gene expression
reference for establishment of the SPORTS mutant rat line: Life Sciences 77 (2005) 551-56; PubMed ID: 15904672

hyb_protocol: 0.6ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized toSurePrint G3 Rat GE 8x60K Microarray (Agilent)）（SurePrint G3 Rat GE 8x60K Microarray (Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).

data_processing:
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
Processed signal intensities were normalized by the global scaling method. A trimmed mean probe intensity was determined by removing 2% of the lower and the higher end of the probe intensities in order to calculate the scaling factor. Normalized signal intensities were then calculated from the target intensity on each array using the scaling factor, so that the trimmed mean target intensity of each array was arbitrarily set to 2500.

organism: Rattus norvegicus

source_name: SPORTS rat, brain

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug Total RNA using the Low Input Quick Amp Labeling Kit(Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.

extract_protocol: RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia CA) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

tag: background strain

value: Wistar

tag: genotype

value: SPORTS

value: brain

tag: gender

value: male

tag: age


title: S7BR


title: NaClrep1


title: MARVMut_24hr_rep2

city: Frederick

first: Catherine

middle: E

last: Arnold

state: MD

address: 1425 Porter St

department: Diagnostic Systems Division

institute: US Army Medical Research Institute of Infectious Diseases

data_processing:
dual index filter: in house software (raw reads index filtered to Q30 cutoff)
trimming : Trimmomatic-0.33 (leading:3, trailing:3, sliding window:4:15, minlen: 80, Q30 cutoff)
alignment and normalization: kallisto 0.43.0 (bootstraps: 100, default parameters)
Genome_build: Raegyp2.0
Supplementary_files_format_and_content: abundance files: h5 (contains transcript_id, length, eff_length (effective length), est_counts (estimated counts) and tpm (transcripts per million) for each gene in the transcriptome, includes bootstrap values (each h5 contains 100 bootstraps that are used for downstream differential gene expression).  length, eff_length, est_counts used to calculate tpm.  )
Supplementary_files_format_and_content: annotation: gff (contains gene identifiers used in h5 files, genome coordinates, Ensembl IDs, gene symbols, etc)

organism: Rousettus aegyptiacus

growth_protocol: 10% FBS DMEM, 5% CO2 and 37C

extract_protocol:
Harvested in 1x RNA Lysis/Binding Solution Concentrate (Thermo Fisher Scientific) at 3hr, 8hr and 24hr post-infection, followed by magnetic bead purification and TURBO DNase treatment using the MagMAX-96 Total RNA Isolation Kit (Ambion) according to manufacturer guidelines. RNA extractions were performed on a MagMAX Express 96 Magnetic Particle Processor (Applied Biosystems)
Libraries were generated on the Sciclone G3 Liquid Handling Robot (PerkinElmer) using the TruSeq Stranded Total RNA Library Prep Kit (Illumina). Library quality was evaluated on the Agilent 2200 TapeStation 2200 and quantified by qPCR using the KAPA Complete (Universal) qPCR kit (Kapa Biosystems) for Illumina libraries. Libraries were diluted to 12 pM and cluster generation was performed on the Illumina cBot. Libraries were sequenced on the Illumina HiSeq 2500 using the paired end 2x125 bp, dual-index format.

treatment_protocol: MOI 1.0 of Sendai (Cantell, Charles River), MARV371bat, MARV371-R301A, or mock (DMEM media alone), media changed afer 1 hour of absorption and cells incubated at 5% CO2 and 37C

tag: cell type

value: RoNi7.1 (fibroblast like kidney derived)

tag: infection

tag: time

tag: passage

value: 4 to 6


title: Mouse 10.5

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_10_5


title: S9BR


title: C7BR

source_name: Wild type, brain

value: wildtype


title: NaHCO3rep2

value: Exposed to NaHCO3


title: C6BR


title: wild-type 37C TSS-seq 2

description: Transcription Start Site Sequencing (TSS-seq) of a wild-type S. cerevisiae strain grown at 30ºC and shifted to 37ºC for 80 minutes.


title: NaHCO3rep1


title: wild-type 37C TSS-seq 1


title: MARVMut_24hr_rep1


title: Mouse 10.3

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_10_2


title: D2 replicate 2

city: Taipei

first: Ya-Ping

last: Yen

address: 128 Academia Road, Section 2, Nankang

department: Institute of molecular biology

country: Taiwan

institute: Academia Sinica

data_processing:
Adapter contamination in the paired-end reads was removed using PEAT.
The trimmed reads were aligned to the mm10 genome with STAR.
GENCODE (version M9) was used to annotated to aid the junction read alignment in STAR the output of which was submitted to Cufflinks for de novo transcript assembly with the option ‘library type; first-strand’ to allow strand-specific alignments.
Only transcripts that were longer than 200 bp, had no overlap with any known genes, and consisted of more than one exon were regarded as novel lncRNAs.
pooled these novel lncRNAs along with all known genes annotated in GENCODE and used HTseq to calculate the read count aligned onto each transcript.
Normalized read counts by TMM (M: 30%, A: 5%)
calculated the specificity score of each transcript among the samples at different stages according to the Jensen–Shannon definition for tissue specificity scores.
Mormalized read count > 10, fold-change > 1.25 and the transcripts were split into three groups: protein coding genes, annotated lncRNAs, and novel lncRNAs.
Genome_build: mm10
Supplementary_files_format_and_content: Paired-end, strand-specific RNA-seq Alignment reads to mouse genome not overlapping with known genes. mm10 transcriptome including novel lncRNAs and known genes. Read count of each transcript tallied by HTSeq. Normalized read counts by TMM (M:30%; A:5%). Stage-specific lncRNAs (normalized read count > 10 ,fold-change > 1.25 )   mm10 by STAR. Novel lncRNAs by cufflinks

organism: Mus musculus

source_name: neural epithelium (NE)

growth_protocol: ESCs were cultured and differentiated into spinal MNs as previously described (Wichterle et al., 2002; Wichterle et al., 2009). Cells were trypsinized and collected for FACS at day 7 to purify GFPon neurons for strand-specific RNA-seq.

extract_protocol:
FACS sorted cells were harvested, flash frozen on dry ice, and RNA was harvested using Trizol reagent.
Illumina TruSeq RNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries. RNA libraries were prepared for sequencing using standard Illumina protocols.

value: neural epithelium (NE)

value: Day 2

value: wild type


title: MARVMut_8hr_rep3

value: RoNi7.1(fibroblast like kidney derived)


title: C5BR


title: MARVMut_8hr_rep2


title: D2 replicate 1


title: Mouse 10.2

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_10_1


title: Mouse 10.1

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_10_0


title: ES_mouse

source_name: embryonic stem cell (ESC)

value: embryonic stem cell (ESC)

value: Day 0


title: Mouse 6.5

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_6_5


title: MARVMut_3hr_rep3


title: Mouse 10.4

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_10_4


title: MARVMut_3hr_rep2


title: MARVMut_3hr_rep1


title: Mouse 6.3

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_6_2


title: Mouse 6.4

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_6_4


title: Mouse 6.2

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_6_1


title: KYSE150 galectin-9 treated 6

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

institute: Toray Industries,Inc.

hyb_protocol: Hybridized for 16 h at 32 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..

scan_protocol: 3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).

data_processing: The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.

organism: Homo sapiens

source_name: cell line galectin-9

label_protocol: Extracted total RNA was labeled with Cy5 using the 3D-Gene miRNA labeling kit (Toray Industries Inc., Tokyo, Japan).

growth_protocol: KYSE150 cells were cultured in Ham's F12 supplemented with 2% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C.

extract_protocol: RNA samples were extracted from cells using miRNesy mini kit (Quiagen).

treatment_protocol: KYSE150 cells were treated with or without 100 nM galectin-9 for 6 h

tag: cell line

value: esophageal squamous cell carcinoma


title: MARVMut_8hr_rep1


title: Mouse 6.1

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_6_0


title: MARV_WT_24hr_rep3


title: KYSE150 galectin-9 treated 5


title: 557287A12 - R108-bacA_R3_bacA vs R108-Sm1021_R3_WT

city: Orsay

first: Stéphanie

last: Pateyron

address: Rue de Noetzlin _ Batiment 630

country: France

institute: IPS2_Institute of Plant Sciences Paris-Saclay

description: rhizobium WT versus BacA (exp2).

hyb_protocol: R108-Sm1021_R3_WT Cy5 / R108-bacA_R3_bacA Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: Mapix, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%

data_processing: For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths Cy5 (red) and Cy3 (green).For each array, a global intensity-dependent normalization using the loess procedure (Yang et al., 2002) was performed to correct the dye bias.Log-ratios are then averaged over the duplicate probes to get a value per gene.

organism: Medicago truncatula

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: aerial - Media: BNM Agar Hygrometry: 100 Température: 26°C

treatment_protocol: no treatment

tag: genotype/variation

tag: ecotype

tag: harvest date

value: BacA mutant


title: 557287A10 - 23_R2_bacA vs 22_R2_WT

description: rhizobium WT versus BacA (exp2).

hyb_protocol: 22_R2_WT Cy5 / 23_R2_bacA Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 557287A11 - R108-Sm1021_R3_WT vs R108-bacA_R3_bacA

description: rhizobium WT versus BacA (exp2).

hyb_protocol: R108-bacA_R3_bacA Cy5 / R108-Sm1021_R3_WT Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 557287A09 - 22_R2_WT vs 23_R2_bacA

description: rhizobium WT versus BacA (exp2).

hyb_protocol: 23_R2_bacA Cy5 / 22_R2_WT Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: MARV_WT_24hr_rep2


title: MARV_WT_24hr_rep1


title: MARV_WT_8hr_rep3


title: Mouse 3.4

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_3_3

value: Mus musculus BALB/c

tag: strain


title: Mouse 3.5

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_3_4


title: KYSE150 galectin-9 treated 3


title: KYSE150 galectin-9 treated 4


title: KYSE150 galectin-9 treated 2


title: Mouse 3.3

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_3_2


title: 557287A08 - 15_R1_bacA vs 7_R1_WT

description: rhizobium WT versus BacA (exp2).

hyb_protocol: 7_R1_WT Cy5 / 15_R1_bacA Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: MARV_WT_8hr_rep2


title: KYSE150 control 6

source_name: cell line control


title: Mouse 3.2

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_3_1


title: KYSE150 control 5


title: KYSE150 galectin-9 treated 1


title: Mouse 3.1

description:
cDNA Amplicon
VSG transcripts were enriched by carrying out PCR with oligonucleotide primers specific to the T. brucei spliced leader sequence (TbSL) and a reverse primers complementary to a 13 base pair conserved region in VSG 3’ untranslated regions (3'UTR)
balbc_3_0


title: MARV_WT_8hr_rep1


title: 557287A06 - 9_R108_3dpi_WSM419_BactoA_rep3 vs 10_R108_3dpi_WSM419_PhytaG_rep3

description: Agar medium versus Phytagel medium (exp1)

hyb_protocol: 10_R108_3dpi_WSM419_PhytaG_rep3 Cy5 / 9_R108_3dpi_WSM419_BactoA_rep3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

growth_protocol: seedling - Media: Buffered nidulation medium. Hygrometry: 100%. Temperature: 26°C.

treatment_protocol: Name:Phytagel medium - compound based treatment - compound addition,agar:quantity 0.8percent time 72hour . Medicago truncatula R108 seedlings were inoculated with Sinorhizobium medicae WSM419 and were cultivated during three days on buffered nidulation medium solidified with Phytagel or Agar.

value: 3 days

tag: treatment

value: Phytagel medium

treatment_protocol: Name:Agar medium - compound based treatment - compound addition,agar:quantity 2percent time 72hour . Medicago truncatula R108 seedlings were inoculated with Sinorhizobium medicae WSM419 and were cultivated during three days on buffered nidulation medium solidified with Phytagel or Agar.

value: Agar medium


title: MARV_WT_3hr_rep3


title: 557287A05 - 10_R108_3dpi_WSM419_PhytaG_rep3 vs 9_R108_3dpi_WSM419_BactoA_rep3

hyb_protocol: 9_R108_3dpi_WSM419_BactoA_rep3 Cy5 / 10_R108_3dpi_WSM419_PhytaG_rep3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: KYSE150 control 4


title: 557287A07 - 7_R1_WT vs 15_R1_bacA

description: rhizobium WT versus BacA (exp2).

hyb_protocol: 15_R1_bacA Cy5 / 7_R1_WT Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 557287A04 - 13_R108_3dpi_WSM419_BactoA_rep2 vs 14_R108_3dpi_WSM419_PhytaG_rep2

hyb_protocol: 14_R108_3dpi_WSM419_PhytaG_rep2 Cy5 / 13_R108_3dpi_WSM419_BactoA_rep2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 557287A03 - 14_R108_3dpi_WSM419_PhytaG_rep2 vs 13_R108_3dpi_WSM419_BactoA_rep2

hyb_protocol: 13_R108_3dpi_WSM419_BactoA_rep2 Cy5 / 14_R108_3dpi_WSM419_PhytaG_rep2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 557287A02 - 11_R108_3dpi_WSM419_BactoA_rep1 vs 12_R108_3dpi_WSM419_PhytaG_rep1

hyb_protocol: 12_R108_3dpi_WSM419_PhytaG_rep1 Cy5 / 11_R108_3dpi_WSM419_BactoA_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: KYSE150 control 3


title: MARV_WT_3hr_rep1


title: wild type transposon library control 1

city: Boulder

first: Jake

middle: James

last: Flood

state: CO

address: 347 UCB

department: Molecular, Cellular and Developmental Biology

institute: University of Colorado Boulder

description: Wildtype control and PCP treated samples are both compared to the same time zero samples

library_source: genomic

data_processing:
Library strategy: Tn-Seq
Bcl2fastq software (v 2.17.1.4) was used for base-calling
Sequenced reads were demultiplexed (fastx_barcode_splitter, 11sep2008) and trimmed from the 3' end to 27bp (fastx_trimmer, v0.0.14).
MmeI cuts at a variable distance (20-21bp) from its recognition site. Therefore, reads were separated by the distance between the transposon and the cut site using fastx_barcode_splitter (11sep2008). Remaining transposon DNA was then trimmed from the read.
Read with a Phred score less than 30 in more than 50% of the read were removed using fastq_quality_filter (v0.0.14).
Reads were mapped to the S. chlorophenolicum L-1 genome (Genbank assembly accession GCA_000147835.3) with bowtie (v1.0.0) using options “--best" and “--tryhard", and a maximum seed mismatch of 1. Reads aligning to multiple places in the genome were discarded, as there is no way to identify which gene has been disrupted.
Samples were then analyzed as described in the MAGenTA workflow (McCoy KM, Antonio ML, van Opijnen T. 2017. MAGenTA - Microbial Assessment by Genome-Wide Tn-Seq Analysis - Manual. Boston College, Chestnut Hill, MA) using default parameters except where described below. Inserts with fewer than 8 reads and inserts occurring in the first or last 5% of the gene were eliminated from the analysis. Inserts that were present before outgrowth (t=0) but were completely absent after outgrowth were assigned one read so that more accurate fitness values could be calculated.
To determine significant fitness differences, we performed a one- or two-sample t-test (where appropriate) with Bonferroni correction. These final results can be found in our paper.
Genome_build: GCA_000147835.3
Supplementary_files_format_and_content: comma separated text files containing normalized read counts for each transposon insertion location in the genome.

molecule: genomic DNA

organism: Sphingobium chlorophenolicum L-1

source_name: liquid culture

growth_protocol: Transposon libraries were grown in SCM medium at 30˚C with shaking in the presence or absence of 200 µM PCP for 22-24 generations. Cells were passaged every 2-3 generations to maintain low cell optical density and constant PCP concentrations. SCD medium (pH 7.1) contains 3.7 mM K2HPO4, 1.4 mM KH2PO4, 0.4 mM MgSO4.7H2O, 5.9 mM NaNO3, 23.7 mM sodium glutamate, 20 µM FeSO4, 90 µM CaCl2, and 0.1x EZ-Rich supplement mixes M2103 and M2104, which contain nucleobases, vitamins, and amino acids with the exception of threonine and leucine.

extract_protocol:
Total genomic DNA was purified from cultures using the Purelink Genomic DNA Mini Kit (Invitrogen, K1820-02).
Tn-Seq libraries were prepared as described in (Wiles TJ, Norton JP, Russell CW, Dalley BK, Fischer KF, Mulvey MA. 2013. Combining quantitative genetic footprinting and trait enrichment analysis to identify fitness determinants of a bacterial pathogen. PLoS Genet 9). Briefly, the transposon and the immediate surrounding genomic DNA were isolated by digestion with MmeI and size selection on an agarose gel. The fragments were then dephosphorylated, ligated to adaptors, and PCR amplified to prepare for Illumina short-read sequencing.

treatment_protocol: See growth protocol

tag: medium

value: SCM medium (see growth protocol)

tag: generations grown

tag: stress


title: MARV_WT_3hr_rep2

value: RoNi7.1 (fibroblast like kidney derived)


title: wild type transposon library control 3


title: wild type transposon library control 2


title: KYSE150 control 2


title: SeV_24hr_rep3

value: Sendai Cantell

value: 4 to 6


title: P3_scars

city: Utrecht

first: Maria

last: Florescu

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

description: scared GFP per organ for one fish

data_processing:
Library strategy: ScarTrace
Library is demultiplexed according to their barcodes (frist 8 nucleotides in first read R1).
Second reads (R2) with a valid cell barcode in first read are mapped to the reference GFP sequence with bwa mem (version bwa 0.7.16a) producing a sam file.
Sam file is checked for mapped reads containing primer sequence used in the PCR (GGCCCCGTGCTGCTGCCCGAC) with a Hamming distance equal or less than 3. Proper reads with scars are counted and saved.
Proper reads with scars from the samfile are remaped using the biopython function pairwise2.align.globalms with match (=1), mismatch (=0.25), open (= -1) and extend parameters (= -0.1)
Genome_build: eGFP sequence extended with ERCC92
Supplementary_files_format_and_content: tabular separated values (in tsv files) per fish including the raw scars counts (shown as cigar code) per organ before filtering and normalization.

organism: Danio rerio

source_name: multiple organs

extract_protocol:
Parts of tissues were dissected from different organs and flash frozen in liquid nitrogen. Genomic DNA was isolated from the body structures using DNeasy Blood & Tissue Kits (Qiagen, Cat No./ID: 69506)
GFP sequences were amplified by PCR with primers complementary to GFP, including an 8 bp barcode sequence and adapter sequences for Illumina sequencing. sequenceable libraries were generated by a second round of PCR with indexed primers from Illumina’s TruSeq Small RNA Sample Prep Kit.

tag: tissue

value: multiple organs

tag: cas9 injection

value: Cas9 protein

value: 1 year

tag: FISH id

tag: molecule subtype

value: Scarred GFP


title: wild type transposon library PCP 3

value: 200 µM PCP

value: wildtype


title: P2_scars

description: scared GFP per organ for one fish

tag: age


title: SeV_24hr_rep2

tag: cell type

tag: infection


title: KYSE150 control 1


title: SeV_24hr_rep1

tag: time

tag: passage


title: 557287A01 - 12_R108_3dpi_WSM419_PhytaG_rep1 vs 11_R108_3dpi_WSM419_BactoA_rep1

hyb_protocol: 11_R108_3dpi_WSM419_BactoA_rep1 Cy5 / 12_R108_3dpi_WSM419_PhytaG_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: P1_scars

description: scared GFP per organ for one fish


title: wild type transposon library PCP 1

tag: genotype/variation


title: C1RM1

description: Gene expression

hyb_protocol: 1.65 ug of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Rat Genome Oligo Microarrays (G4858A) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44K array slides (Scan Area 61x21.6 mm, Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028282_D_F_20110531) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: Wild type, muscle

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

value: skeletal muscle

tag: gender

value: male


title: wild type transposon library PCP 2


title: R3_scars

description: scared GFP per organ for one fish

value: Cas9 RNA


title: SeV_8hr_rep3


title: 563100A08 - Nodules_controle_rep1 vs Roots_controle_rep1

description: which are the genes differentially expressed in alfalfa spontaneous (non rhizobium-infected) nodules vs. control roots?

hyb_protocol: Roots_controle_rep1 Cy5 / Nodules_controle_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

organism: Medicago sativa

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, total RNA  .

growth_protocol: roots - Media: Caisson growth medium without nitrogen Hygrometry: 75% Temperature: 21°C Light: 350 µmol/m²/s

value: roots

growth_protocol: nodule - Media: Caisson growth medium without nitrogen Hygrometry: 75% Temperature: 21°C Light: 350 µmol/m²/s

value: nodules


title: 563100A06 - Nodules3_rep3 vs Roots3_rep3

hyb_protocol: Roots3_rep3 Cy5 / Nodules3_rep3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

growth_protocol: roots - Media: Caisson growth medium without nitrogen (Cuttings were grown with caisson medium (as described in the Medicago truncatula handbook) supplemented with 5mM of NH4NO3 during one week. Then they were starved for nitrogen.) Hygrometry: 75% Temperature: 21°C Light: 350 µmol/m²/s

extract_protocol: Roots3_rep3:6ng. (Qiagen_RNeasy.pdf)

treatment_protocol: Name:Nitrogen deficiency_3 days - environmental treatment - nitrogen concentration,nitrogen:time 3day .

growth_protocol: nodule - Media: Caisson growth medium without nitrogen (Cuttings were grown with caisson medium (as described in the Medicago truncatula handbook) supplemented with 5mM of NH4NO3 during one week. Then they were starved for nitrogen.) Hygrometry: 75% Temperature: 21°C Light: 350 µmol/m²/s

extract_protocol: Nodules3_rep3:6ng. (Qiagen_RNeasy.pdf)

treatment_protocol: Name:Nitrogen deficiency_19 days - environmental treatment - nitrogen concentration,nitrogen:time 19day .


title: 563100A07 - Roots_controle_rep1 vs Nodules_controle_rep1

hyb_protocol: Nodules_controle_rep1 Cy5 / Roots_controle_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 563100A05 - Roots3_rep3 vs Nodules3_rep3

hyb_protocol: Nodules3_rep3 Cy5 / Roots3_rep3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: wild type transposon library time zero 3


title: wild type transposon library time zero 2


title: SeV_8hr_rep2


title: SeV_8hr_rep1


title: R1_scars

description: scared GFP per organ for one fish


title: Null Rep A

city: Nashville

first: Danyvid

last: Olivares-Villagomez

state: TN

address: 21 Ave and Garland, 811 Light Hall

institute: Vanderbilt University

data_processing:
Sequenced reads were analyzed using the transcript quantification tool Salmon
Transcripts Per Million (TPM) values were used for all downstream analysis
Supplementary_files_format_and_content: tab delimited txt (.sf.txt) files include num reads, length, and tpm values for each transcript

source_name: Intraepithelial Lymphocytes RNA

extract_protocol:
RNA was harvested using the Rneasy Micro Kit (Cat# 74004)
RNA libraries were prepared for sequencing using standard stranded Illumina protocols

treatment_protocol: CD45+ FACs sorting IEL were incubated in RPMI + 10% serum. The osteopontin treated cells were incubated with 2ug/ml osteopontin. Both osteopontin treated and null treated cells were incubated for 24hrs at 37 C

value: CD45+ Intraepithelial Lymphocytes


title: SeV_3hr_rep2


title: R2_scars

description: scared GFP per organ for one fish


title: ∆pcpB transposon library control 3

description: pcpB control and PCP treated samples are both compared to the same time zero samples

value: pcpB


title: Null Rep 2


title: Null Rep B


title: 563100A03 - Roots2_rep2 vs Nodules2_rep2

hyb_protocol: Nodules2_rep2 Cy5 / Roots2_rep2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: Nodules2_rep2:6ng. (Qiagen_RNeasy.pdf)

extract_protocol: Roots2_rep2:6ng. (Qiagen_RNeasy.pdf)


title: wild type transposon library time zero 1

value: SCM medium (see growth protocol)


title: Null Rep 1


title: ∆pcpB transposon library control 2


title: SeV_3hr_rep1

value: RoNi7.1 (fibroblast like kidney derived)


title: SeV_3hr_rep3

value: Sendai Cantell


title: 563100A01 - Roots1_rep1 vs Nodules1_rep1

hyb_protocol: Nodules1_rep1 Cy5 / Roots1_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

extract_protocol: Nodules1_rep1:6ng. (Qiagen_RNeasy.pdf)

extract_protocol: Roots1_rep1:6ng. (Qiagen_RNeasy.pdf)


title: 563100A04 - Nodules2_rep2 vs Roots2_rep2

hyb_protocol: Roots2_rep2 Cy5 / Nodules2_rep2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: ∆pcpB transposon library control 1


title: Opn Rep A


title: 563100A02 - Nodules1_rep1 vs Roots1_rep1

hyb_protocol: Roots1_rep1 Cy5 / Nodules1_rep1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: Opn Rep 2


title: ∆pcpB transposon library PCP 3


title: ∆pcpB transposon library PCP 1


title: Opn Rep 1


title: ∆pcpB transposon library PCP 2


title: ∆pcpB transposon library time zero 3


title: Opn Rep B


title: ∆pcpB transposon library time zero 1


title: ∆pcpB transposon library time zero 2


title: PBEC_Dex-and-TNFa_ANR273_rep3

city: Hershey

first: Faoud

middle: T

last: Ishmael

state: PA

address: 500 University Dr.

department: Division of Pulmonary and Critical Care Medicine

institute: Penn State College of Medicine

description:
Array D, blocks 25-32 of raw data file
2013-03-1_19717741_650PMT_5um_0532.txt

hyb_protocol: Labeled LMW RNA samples were hybridized to the MicroRNA microarrays according to conditions recommended in the Flash Tag RNA labeling Kit manual.

scan_protocol: The microarrays were scanned on an Axon Genepix 4000B scanner, and data was extracted from images using GenePix V4.1 software.

data_processing: Data for 4 arrays were extracted from a single microarray image into a single text file using GenePix version 4.1 software. The raw text files were split in to four subfiles based on block assignment, namely blocks 1-8 contained data for array A, blocks 9-16 contained data for array B, blocks 17-24 contained data for array C, and blocks 25-32 contained data for array D. Spot intensities were obtained for the 10336 features on each microarray by subtracting the median local background from the median local foreground for each spot. Detection Thresholds for each array were determined by calculating the 10% trim mean intensity of the negative controls spots and adding 5X the standard deviation of the background (non-spot area). The spot intensities and the Threshold (T) were transformed by taking the log (base 2) of each value.  The mean probe intensities for each of the 2017 human probes on each of the 12 arrays were then determined by averaging the triplicate spot intensities. Spots flagged as poor quality during data extraction were omitted prior to averaging and spots that have an average saturated signal were removed prior to normalization, which yielded 1995 human non-saturated probes intensities. The normalization factor (N) for each microarray was determined by obtaining the 20% trim mean of the mouse probes intensities above Threshold in > 90% of samples. The log2-transformed spot intensities for all 10,336 features were normalized, by subtracting N from each spot intensity, and scaled by adding the grand mean of N across all microarrays.  The 1995 human non-control non-saturated log2-transformed, normalized, and averaged probe intensities were filtered to obtain a list of 606 human microRNA probes showing probe intensity above Threshold in at least 10% of samples. The data was untransformed using the formula 2^N.

source_name: NuLi-1 human airway epithelial cell line, Dex+TNFa treated

label_protocol: The total RNA was DNAse digested and low-molecular weight (LMW) RNA was isolated by ultrafiltration through YM-100 columns (Millipore) and subsequent purification using the RNeasy MinElute Clean-Up Kit (Qiagen). The LMW RNA samples were 3’-end labeled with Oyster-550 fluorescent dye using the Flash Tag RNA labeling Kit (Genisphere).

extract_protocol: RNA was extracted using the TRIzol method

tag: glucocorticoid treatment

value: Dex

tag: cytokine treatment

value: TNFa


title: MARVMut_24hr_rep3

value: 4 to 6


title: PBEC_Dex-and-TNFa_ANR268_rep2

description:
Array D, blocks 25-32 of raw data file
2013-03-1_19717740_650PMT_5um_0532.txt


title: pentachlorophenol_5hour_rnaseq 2

description: 200 µM pentachlorophenol

data_processing:
Bcl2fastq software (v 2.17.1.4) was used for base-calling
Sequenced reads were demultiplexed (fastx_barcode_splitter, 11sep2008) and trimmed of adaptor (fastx_trimmer, v0.0.14).
Processed reads were mapped to the S. chlorophenolicum genome (GCA_000147835.3, NCBI) with bowtie2 (v2.1.0) with default parameters.
Counts per gene were obtained using HTSeq (v0.7.2).
Genome_build: GCA_000147835.3
Supplementary_files_format_and_content: tab delimited text files containing read counts for all protein-coding regions of the genome

growth_protocol: All cells were grown in 1/4-strength tryptic soy broth at 30˚C to an optical density (600 nm) of 0.3-0.4. Stressor was added and cells were incubated with shaking at 30˚C for 15 minutes. Cells were then harvested for total RNA extraction.

extract_protocol:
Total RNA was purified using Ambion Purelink RNA mini kit according to manufacturer's instructions. Cells were homogenized using Qiagen Qiashredder columns. RNA was eluted in Rnase-free water. After elution, samples were treated with Turbo DNA-free kit (Ambion).
RNAtag-seq libraries were prepared as described in (Shishkin, Alexander A., et al. "Simultaneous generation of many RNA-seq libraries in a single reaction." Nature methods 12.4 (2015): 323.).

treatment_protocol: 2 mL of culture was immediate mixed with Qiagen RNAProtect Bacterial Reagent. Tubes were incubated at room temperature for 5 minutes, then spun at 4 x g at 4˚C for 10 minutes. The supernatent was removed and cell pellet was frozen and stored at -70˚C.

value: 1/4-strength tryptic soy broth

tag: exposure time

value: 5 hours

value: 200 microM pentachlorophenol


title: pentachlorophenol_5hour_rnaseq 3

tag: medium


title: PBEC_Dex-and-TNFa_ANR267_rep1

description:
Array D, blocks 25-32 of raw data file
2013-03-1_19717739_650PMT_5um_532_redip.txt


title: PBEC_TNFa_ANR273_rep3

description:
Array D, blocks 25-32 of raw data file
2013-03-7_19717751_650PMT_5um_0532.txt

source_name: NuLi-1 human airway epithelial cell line, TNFa treated

value: control


title: PBEC_TNFa_ANR267_rep2

description:
Array B, blocks 9-16 of raw data file
2013-03-7_19717751_650PMT_5um_0532.txt


title: pentachlorophenol_5hour_rnaseq 1

description: 200 µM pentachlorophenol


title: PBEC_TNFa_ANR268_rep1

description:
Array C, blocks 17-24 of raw data file
2013-03-1_19717740_650PMT_5um_0532.txt


title: Fry (alevins) originating from females held at 17°C during the periovulatory period [28-17 YSR 1a]

city: Rennes

first: Aurelie

last: LE CAM

address: Campus de Beaulieu

department: PHASE

institute: INRA

hyb_protocol: 600 ng of Cy3-cRNA was fragmented and hybridized on a sub-array. Hybridization was carried out for 17 hours at 65°C in a rotating hybridization oven prior to washing.

scan_protocol: Microarrays were scanned with an Agilent Scanner (Agilent DNA Microarray Scanner, Agilent Technologies, Massy, France) using the standard parameters for a gene expression 8x60K oligoarray (3µm and 20bits)

data_processing: Data were then obtained with the Agilent Feature Extraction software (10.7.3.1) according to the appropriate GE protocol (GE1_107_Sep09). Normalization and statistical analysis were performed using GeneSpring software (Agilent).

organism: Oncorhynchus mykiss

label_protocol: Labeling and hybridization steps were performed following the “One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling)”Agilent protocol. Briefly, for each sample, 150 ng of total RNA was amplified and labeled using Cy3-CTP. Yield (>0.825µg cRNA) and specific activity (> 6pmol of Cy3 per µg of cRNA) of Cy3-cRNA produced were checked with the Nanodrop.

growth_protocol: Two-year old females rainbow trout were exposed to either 12°C (12°C group, standard reproduction conditions) or 17°C (17°C group, high suboptimal temperature) for six weeks before ovulation. The temperature of 17°C was selected because it is known to induce a dramatic decrease in embryonic survival {Aegerter, 2004 #2630}. For each group, 30 marked (external tag placed on the dorsal fin) females were kept in 2.5 m3 tanks (2 x 2 x 0.62 m, length × width × water height). In the 17°C group, females initially reared at 12°C were acclimated for five days to an increase of 1°C/day until 17°C. For three weeks preceding ovulation, females were checked every two-three days to detect ovulation. In both experimental group, eggs originating from four simultaneously ovulating females of each group were collected and fertilized using a pool of sperm collected from four males held at 12°C. Fertilization was performed immediately in both groups in order to avoid any bias on subsequent behavioural phenotypes that would have been induced by differences during embryo development. For each of the eight females, fertilizations of 800 eggs were performed at 10°C using the medium ActiFish (IMV, L'Aigle, France; 100 ml ActiFish + 400 ml water) and fertilized eggs were distributed within a tray (20 x 50 cm) in two incubators (10 x 10 cm) (approximately 400 eggs/incubator and two incubators/tray) supplied with 10°C flow-though recycled water. Each tray was covered with a lid to avoid exposure to light.

extract_protocol: RNA was extracted from 20 eggs sampled at fertilization, 20 eggs at 5 dpf, 6 embryos at hatching and 6 alevins sampled at YSR. Frozen tissues were lysed with Precellys Evolution Homogenizer (Ozyme, bertin technologies) in TRI Reagent (TR118, Euromedex) and total RNA was extracted according to the reagent’s method followed by a Nucleospin RNA isolation kit (740955, Macherey Nagel).

treatment_protocol: Developmental success was monitored at eyeing stage, i.e. 19 days post-fertilization (dpf), hatching (32-33 dpf), and completion of yolk-sac resorption (YSR, 55 dpf) by counting dead embryos that were removed from incubators. In both experimental groups, and in all egg clutches, biological samples were collected at four different stages: unfertilized eggs, around zygotic genome activation (i.e. 5 days post fertilization), hatching after removing yolk-sac, and YSR, which also corresponded to the stage of behavioural phenotyping. Entire (i.e. whole body) embryos were sampled. All samples were frozen in liquid nitrogen and held at -80°C until further processing.

tag: developmental stage

value: Yolk-Sac Resorption

tag: growth temperature


title: Fry (alevins) originating from females held at 17°C during the periovulatory period [31-17 YSR 4a]


title: Fry (alevins) originating from females held at 17°C during the periovulatory period [30-17 YSR 3a]


title: methylglyoxal_15min_rnaseq 3

description: 200 µM pentachlorophenol

value: 15 minutes

tag: stress

value: 1 mM methylglyoxal


title: Fry (alevins) originating from females held at 17°C during the periovulatory period [29-17 YSR 2a]


title: Fry (alevins) originating from females held at 12°C during the periovulatory period [25-12 YSR 2a]


title: PBEC_Dex_ANR273_rep3

description:
Array B, blocks 9-16 of raw data file
2013-03-1_19717741_650PMT_5um_0532.txt

source_name: NuLi-1 human airway epithelial cell line, Dex treated


title: Embryos originating from females held at 17°C during the periovulatory period [23-17 HWB 3a]

value: Hatching


title: Fry (alevins) originating from females held at 12°C during the periovulatory period [27-12 YSR 4a]


title: methylglyoxal_15min_rnaseq 2

description: 1 mM methylglyoxal


title: Embryos originating from females held at 17°C during the periovulatory period [22-17 HWB 2a]


title: paraquat_15min_rnaseq 3

description: 1 mM methylglyoxal

value: 20 microM paraquat


title: Fry (alevins) originating from females held at 12°C during the periovulatory period [26-12 YSR 3a]


title: PBEC_Dex_ANR267_rep1

description:
Array B, blocks 9-16 of raw data file
2013-03-1_19717739_650PMT_5um_0532.txt


title: Embryos originating from females held at 17°C during the periovulatory period [21-17 HWB 1a]


title: PBEC_Control_ANR273_rep3

description:
Array C, blocks 17-24 of raw data file
2013-03-7_19717751_650PMT_5um_0532.txt

source_name: NuLi-1 human airway epithelial cell line, control treated


title: paraquat_15min_rnaseq 1

description: 20 µM paraquat


title: CCCP_15min_rnaseq 3

description: 20 µM paraquat

value: 39.1 microM CCCP


title: paraquat_15min_rnaseq 2

description: 20 µM paraquat


title: PBEC_Dex_ANR268_rep2

description:
Array B, blocks 9-16 of raw data file
2013-03-1_19717740_650PMT_5um_0532.txt


title: methylglyoxal_15min_rnaseq 1

description: 1 mM methylglyoxal


title: CCCP_15min_rnaseq 2

description: 39.1 µM CCCP


title: CCCP_15min_rnaseq 1

description: 39.1 µM CCCP


title: toluene_15min_rnaseq 2

description: 5 mM toluene

value: 5 mM toluene


title: toluene_15min_rnaseq 1

description: 5 mM toluene


title: PBEC_Control_ANR267_rep1

description:
Array A, blocks 1-8 of raw data file
2013-03-1_19717739_650PMT_5um_0532.txt


title: PBEC_Control_ANR268_rep2

description:
Array A, blocks 1-8 of raw data file
2013-03-1_19717740_650PMT_5um_532_redip.txt


title: pentachlorophenol_15min_rnaseq 3

description: 5 mM toluene


title: pentachlorophenol_15min_rnaseq 2

description: 200 µM pentachlorophenol


title: pentachlorophenol_15min_rnaseq 1

description: 200 µM pentachlorophenol


title: Embryos originating from females held at 12°C during the periovulatory period [18-12 HWB 2a]


title: Embryos originating from females held at 12°C during the periovulatory period [20-12 HWB 4a]


title: Embryos originating from females held at 17°C during the periovulatory period [15-17 ZGA 3a]

value: 5days post fertilization


title: control_15min_rnaseq 3

description: 200 µM pentachlorophenol


title: Embryos originating from females held at 17°C during the periovulatory period [14-17 ZGA 2a]


title: Embryos originating from females held at 12°C during the periovulatory period [12-12 ZGA 4a]


title: toluene_15min_rnaseq 3

description: 39.1 µM CCCP


title: Embryos originating from females held at 17°C during the periovulatory period [16-17 ZGA 4a]


title: Embryos originating from females held at 12°C during the periovulatory period [17-12 HWB 1a]


title: Embryos originating from females held at 12°C during the periovulatory period [19-12 HWB 3a]


title: Embryos originating from females held at 12°C during the periovulatory period [9-12 ZGA 1a]


title: Embryos originating from females held at 12°C during the periovulatory period [10-12 ZGA 2a]


title: Embryos originating from females held at 17°C during the periovulatory period [13-17 ZGA 1a]


title: Kidnay sample 2

city: Honolulu

first: Pietro

last: Bertino

state: HI

address: 651 Ilalo St., BSB222

department: Cell and Molecular Biology

institute: University of Hawaii

description: Gene expression data

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Clariom S Mouse Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: RNA expression in the different tissues was evaluated using the Clariom S Mouse Array (Affymetrix).

data_processing: Expression values were normalized and summarized into transcript clusters for analysis using Robust Multi-array Average approach in Array Studio (OmicSoft, Cary, NC).

label: biotin

source_name: normal kidney tissue

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: BALB/c mice were injected subcutaneously with 100,000 of either CRH5 or EOH6 MM cells. When tumors reached 100 mm3 they were excised and total RNA extracted. At the same time, RNA was isolated from lungs and kidney excised from naïve BALB/c mice.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Mice were not treated.

tag: Sex

value: female

tag: strain

value: BALB/c


title: Kidney sample 1

description: Gene expression data


title: Eggs originating from females held at 17°C during the periovulatory period [8-17 EGG 4a]

value: Unfertilized Egg


title: EOH6 sample 3

description: Gene expression data

source_name: tumor from EOH6 cells


title: Lung sample 1

description: Gene expression data

source_name: normal lung tissue


title: control_15min_rnaseq 1


title: Embryos originating from females held at 12°C during the periovulatory period [11-12 ZGA 3a]


title: Eggs originating from females held at 17°C during the periovulatory period [6-17 EGG 2a]


title: CC3

city: Geneva

first: Thilo

last: Kohler

address: 1, rue Michel Servet

department: Microbiology and Molecular Medicine

country: Switzerland

institute: University of Geneva

data_processing:
The reads were mapped with the TopHat v.2 software to the Pseudomonas aeruginosa PA14 RefSeq genome NC_008463.1 for the PM samples, the Staphylococcus aureus Newman RefSeq genome NC_009641.1 for the SM samples and a merge of the two for the CC samples
The table of counts with the number of reads mapping to each gene feature of to the Pseudomonas aeruginosa PA14 RefSeq genome NC_008463.1 for the PM samples, the Staphylococcus aureus Newman RefSeq genome NC_009641.1 for the SM samples and a merge of the two for the CC samples was prepared with HTSeq v0.5.3p9
The differential expression analysis was performed with the statistical analysis R/Bioconductor package EdgeR v. 3.4.2.
The genes having a count above 1 count per million reads (cpm) in at least 3 samples were kept for the analysis. The differentially expressed genes tests were done with the exactTest() function using the exact negative binomial test.
Genome_build: PA14 RefSeq genome NC_008463.1 for the PM samples, the Staphylococcus aureus Newman RefSeq genome NC_009641.1 for the SM samples and a merge of the two for the CC samples
Supplementary_files_format_and_content: CPM

organism:
Pseudomonas aeruginosa
Staphylococcus aureus

source_name: Coculture replicate 3

growth_protocol: M14 medium

extract_protocol:
RNA was extracted after 3 h of co-culture
RNA were ribodepleted with the Ribo-Zero rRNA Removal kit (Bacteria) from Illumina (formely Epicentre) with 1300 ng as input material. Libraries were prepared with the Illumina TruSeq Stranded Total RNA kit according to manufacturer's specifications.
Ribo-depleted RNA-seq

treatment_protocol: RNA stabilized in Bacteria Protect reagent (Qiagen)

tag: strains

tag: treatment

value: 3h co-culture in M14 medium


title: Eggs originating from females held at 17°C during the periovulatory period [7-17 EGG 3a]


title: control_15min_rnaseq 2


title: CC1

source_name: Coculture replicate 1


title: mouse cauda sperm

status: Public on Oct 31 2018

first: Chen

last: Chu

address: 450 Brookline Ave, Smith 914

institute: Dana-Farber Cancer Institute

description: 18 - 45 nt

data_processing:
FASTX-Toolkit were used for sequence files preprocessing.
Processed sequence reads were mapped to the mouse mm9 or rat rn5 genome using bowtie (version 0.12.7) with settings -k 1 -v 2.
Genome_build: mm9 for mouse; rn5 for rat
Supplementary_files_format_and_content: uniq_read_count

source_name: spermatozoa

growth_protocol: Normal growth

extract_protocol:
Total RNAs were extracted using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols, and 18 - 45 nt fraction of small RNAs were subjected to library construction.

treatment_protocol: Sperm

tag: cell type

value: Cauda epididymal sperm (purified)

tag: age

value: 2 months


title: Feather dorsal E7 input

city: Los Angeles

first: Randall

middle: B.

last: Widelitz

state: California

address: 2011 Zonal Ave HMR 305

department: Department of Pathology

institute: University of Southern California

data_processing:
Alignment of reads (Bowtie2, UCSC galGal4) was done in Partek Flow
Only unique reads are used for peak calling
QuEST was used for peak calling
The replicates of each sample group are combined to perform peak calling.
Genome_build: Galgal4
Supplementary_files_format_and_content: wig

organism: Gallus gallus

source_name: dorsal skin

growth_protocol: fertilized eggs

extract_protocol:
CHIP
NEBNext® Ultra™ DNA Library Prep Kit

treatment_protocol: untreated

tag: tissue

value: skin

tag: chip antibody


title: Feather dorsal E9 H3K4me3 ChIP-seq rep2


title: Feather dorsal E9 input


title: Feather dorsal E9 H3K4me3 ChIP-seq rep1


title: mouse caput sperm

description: 18 - 45 nt small RNAs

data_processing:
FASTX-Toolkit were used for sequence files preprocessing.
Processed sequence reads were mapped to the mouse mm9 or rat rn5 genome using bowtie (version 0.12.7) with settings -k 1 -v 2.
Genome_build: mm9 for mouse; rn5 for rat

value: Caput epididymal sperm (purified)


title: CRH5 sample 3

description: Gene expression data

source_name: tumor from CRH5 cells


title: CRH5 sample 1

description: Gene expression data


title: Eggs originating from females held at 12°C during the periovulatory period [1-12 EGG 1a]


title: rat cauda sperm

description: 18 - 45 nt

value: Sprague Dawley

value: 450g young adult


title: Feather dorsal E7 H3K4me3 ChIP-seq rep1


title: EOH6 sample 2

description: Gene expression data


title: RRBS_CAID_3_p14

city: Montréal

first: Gregor

middle: Ulrich

last: Andelfinger

state: QC

address: 3175, Chemin de la Côte-Sainte-Catherine

department: Cardiology

country: Canada

institute: CHU Sainte-Justine

data_processing:
Bsmap was used for initial data processing, methylation calls ans reads alignment.
Methylkit was used to do analysis and statistic of differentially methylated regions, specific parameters: 100 bp step-wise tiling windows, 1 CpG minimum per tile and a minimum 20x CpG coverage of each tile per sample.
Significant DNA methylation changes ≥ 30%.
HOMER was used for sequences annotations.
HOMER was used to analyze DNA binding motifs
Genome_build: hg19
Supplementary_files_format_and_content: .map : file containing raw methylation calls. There is one file per sample.

source_name: Human dermal fibroblasts

growth_protocol: Cells were cultured in DMEM, 10% FBS, 1% pen/strep

extract_protocol:
DNA from human fibroblast was extracted using QIAamp DNA mini kit according to the manufacturer's instructions
500ng/sample of extracted DNA was digested with Msp1 enzyme (NEB #R0106M) Methylated adaptors (NEB #E7535L) were ligated with T4 ligase (NEB #M0202M) following by qPCR amplification with Illumina indexing primers. Libraries clean up and size selection was done using Agencourt® AMPure® XP magnetic beads (Beckman Coulter # A63881).

value: Dermal fibroblasts

tag: genotype

tag: passage


title: CRH5 sample 2

description: Gene expression data


title: EOH6 sample 1

description: Gene expression data


title: CC2

source_name: Coculture replicate 2


title: Lung sample 2

description: Gene expression data


title: Eggs originating from females held at 12°C during the periovulatory period [3-12 EGG 3b]


title: Eggs originating from females held at 17°C during the periovulatory period [5-17 EGG 1b]


title: RRBS_CAID_2_p14


title: RRBS_CAID_1_p14


title: RRBS_control_3_p14

value: wild type


title: RRBS_control_1_p14


title: Eggs originating from females held at 12°C during the periovulatory period [2-12 EGG 2a]


title: Eggs originating from females held at 12°C during the periovulatory period [4-12 EGG 4b]


title: RRBS_CAID_3_p8


title: RRBS_control_2_p14


title: RRBS_CAID_2_p8


title: Feather dorsal E7 H3K4me3 ChIP-seq rep2


title: RRBS_control_1_p8


title: RRBS_control_2_p8


title: BR.repo.glia.Pc_DamIDSeq

city: Amsterdam

first: Bas

last: van Steensel

address: Plesmanlaan 121

department: division of Molecular Biology

institute: Netherlands Cancer Institute

data_processing:
Basecalling and filtering were performed using standard software of the Illumina HiSeq 2000.
The 51bp reads from each sample were first adapter clipped and aligned to the reference genome (BDGP R5/dm3) using “bowtie2” (Langmead and Salzberg, Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4),357-9, 2012). For biological samples which were generated using multiple technical replicates (uninduced Dam only controls from larval central brain), the read counts were summed over the technical replicates. Next, reads that did not align or aligned more than once were discarded, as well as the chromosomes “U”, “M” and “Uextra” were excluded from the subsequent analysis. Then, reads were counted by “HTSeq-count” software (Anders et al. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2), 166-9, 2015) in the 1-kb genomic bins and read counts were merged between biological replicates of the same combination of protein / tissue or cell type (Dam only, Dam-Pc, Dam-Lam and Dam-HP1 / larval central brain, Repo+ glial cells of larval central brain, Elav+ neurons of larval central brain and larval fat bodies), as they were highly correlated. After that, the obtained counts were converted to reads per million mapped reads (RPM). Finally, the normalized DamID scores were calculated as the log2 of the ratio of the Dam-Pc/Dam only, Dam-Lam/Dam only and Dam-HP1a/Dam only.
Genome_build: dm3
Supplementary_files_format_and_content: bedgraph format; log2(Dam-POI/Dam)

organism: Drosophila melanogaster

source_name: brain without ventral nerve cord

growth_protocol: Flies were raised at 25°C on standard cornmeal/molasses/agar medium.

extract_protocol:
Two μl of Proteinase K (20 mg/ml) [Roche, 03115887001] were added to dissected central brains and the sample was incubated at 65°C for 6 hours. Next, 1 μl of RNase A (100 mg/ml) [QIAGEN, 1007885] was added and the sample was incubate at 37°C for 30 minutes. The lysate was transferred to a pre-spun (1 minute at 14,000 rpm just prior to use) phase lock gel light 1.5ml tube [5prime, 2302800] and 100 μl of phenol:chloroform:isoamylalcohol (25:24:1) [Sigma-Aldrich, P2069] were added. The sample was mixed well by shaking and the tube was spun for 5 minutes at 14,000 rpm at room temperature. The upper phase (~90 μl) was transferred into a new tube. To precipitate DNA, 1 µl of glycogen (20 mg/ml) [Fermentas, R0561], 10 µl of 3M NaAc (pH 5.2) and 300 µl of ethanol were added, mixed well by inversion and the solution was kept overnight at -20°C. The tube was spun for 30 minutes at 14,000 rpm at 4°C. The supernatant was removed and the pellet was washed with 500 µl of ice cold 70% ethanol. The tube was spun for 5 minutes at 14,000 rpm at 4°C. The supernatant was carefully removed and the pellet was air dried for 15 minutes and dissolved in 12 µl of double distilled water. DNA concentration was measured using a NanoDrop spectrophotometer. Dam-methylated fragments were amplified as described previously (Vogel, M.J. et al. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc 2(6), 1467-78, 2007) using 500 ng of DNA as an input.
The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (QIAGEN, 28104) according to the manufacturer’s instructions. DNA was eluted in nuclease-free water (Ambion, AM9938). From 200 ng to 3 µg of the purified PCR products were diluted in 100 μl of nuclease-free water and transferred into a snap-cap microTUBE (6 × 16 mm) with AFA fiber (Covaris, 520045). DNA was sheared using the Covaris S2 instrument at the following settings: duty cycle = 10%, intensity = 5, cycles/burst = 200, time = 45 sec (as consequence, power = ~23W and bath temperature = ~4°C). The size of the DNA fragments before shearing was usually a smear in the range of 250–2000 bp with a peak at 600 bp, after shearing this was reduced to become a range of 100–500 bp with a peak around 300 bp. After shearing, the DNA was purified and concentrated using Agencourt magnetic beads (Beckman Coulter, A63881; 160 µl of beads were added to 100 µl of sheared DNA solution). DNA was eluted in 50 µl of nuclease-free water. DNA concentration was measured using an Agilent BioAnalyzer 2100 DNA 7500 chip (Agilent, 5067-1506). One μg of sheared material was used to prepare Illumina sequencing library with TruSeq DNA HT Sample Prep Kit (Illumina, FC-121-2003) according to the manufacturer’s instructions. Seven cycles of PCR were performed. This step included sample indexing. The library was quantified on an Agilent BioAnalyzer 2100 DNA 7500 chip that lists the product size distribution and quantifies the concentration in µg/µl and in nM.

treatment_protocol: Central brains were dissected from 20 larvae in 1×PBS [140 mM NaCl, 10 mM PO4 Buffer, 3 mM KCl; Invitrogen cat. no. 18912-014] using forceps and dissection needles and collected in 100 μl TENS solution [100 mM Tris-HCl (pH 8.0); 5 mM EDTA (pH 8.0); 200 mM NaCl; 0.2% SDS] at room temperature.

tag: gender

value: pooled male and female

value: wandering third instar larvae

value: brain without ventral nerve cord

tag: dam-fusion protein

value: Dam-Polycomb

tag: damid system used

value: FLP-inducible STOP#1-Dam


title: BR.elav.neurons.Dam_DamIDSeq

value: Elav+ neurons of larval central brain

value: P{UAS-FLP.Exel}1, y,w/+; M{w[+mC]=hs.min(FRT.STOP1)dam}ZH-51C/+; P(GAL4-elav.L)3/+

value: Dam only


title: BR.repo.glia.Lam_DamIDSeq

value: Repo+ glial cells of larval central brain

value: Dam-Lamin Dm0


title: BR.elav.neurons.Lam_DamIDSeq

value: P{UAS-FLP.Exel}1, y,w/+;  M{w[+mC]=hs.min(FRT.STOP1)dam-Lam}ZH-51C/+; P(GAL4-elav.L)3/+


title: BR.elav.neurons.Pc_DamIDSeq

value: P{UAS-FLP.Exel}1, y,w/+; M{w[+mC]=hs.min(FRT.STOP1)dam-Pc}ZH-51C/+; P(GAL4-elav.L)3/+


title: BR.elav.neurons.HP1a_DamIDSeq

value: P{UAS-FLP.Exel}1, y,w/+; M{w[+mC]=hs.min(FRT.STOP1)dam-HP1}ZH-51C/+; P(GAL4-elav.L)3/+


title: BR.repo.glia.HP1a_DamIDSeq


title: BR.Lam.m_DamIDSeq

value: larval central brain

value: male

value: Dam4-HT-intein@L127C, uninduced


title: RRBS_control_3_p8

value: Dermal fibroblasts

value: wild type


title: FB.Dam.m_DamIDSeq

source_name: fat bodies

growth_protocol: Flies were raised at 25°C on standard cornmeal/molasses/agar medium supplemented with 4-hydroxytamoxifen (Sigma-Aldrich, H7904) at a final concentration of 25 µM.

extract_protocol:
Four hundred μl of Lysis Buffer [5% sucrose; 100 mM Tris-HCl (pH 9.1); 50 mM EDTA (pH 8.0); 5% SDS] and 5 μl of Proteinase K (20 mg/ml) [Roche, 03115887001] were added to dissected fat bodies, the tissue was disrupted by passing through 21G needle 15-20 times and the sample was incubated at 55°C with 600 rpm shaking for 4 hours. Next, 2 μl of RNase A (100 mg/ml) [QIAGEN, 1007885] were added and the sample was incubate at 37°C for 30 minutes. The lysate was transferred to a pre-spun (1 minute at 14,000 rpm just prior to use) phase lock gel heavy 1.5ml tube [5prime, 2302810] and 500 μl of phenol:chloroform:isoamylalcohol (25:24:1) [Sigma-Aldrich, P2069] were added. The sample was mixed well by shaking and the tube was spun for 5 minutes at 14,000 rpm at room temperature. The upper phase was transferred to another pre-spun phase lock gel heavy 1.5ml tube. Five hundred μl of phenol:chloroform:isoamylalcohol (25:24:1) [Sigma-Aldrich, P2069] were added, mixed well by shaking and the tube was spun for 5 minutes at 14,000 rpm at room temperature. The upper phase (~400 μl) was transferred into a new tube. To precipitate DNA, 1 µl of glycogen (20 mg/ml) [Fermentas, R0561], 40 µl of 5M NaCl and 1 ml of ethanol were added, mixed well by inversion and the solution was kept overnight at -20°C. The tube was spun for 1 hour at 14,000 rpm at 4°C. The supernatant was removed and the pellet was washed with 500 µl of ice cold 70% ethanol. The tube was spun for 15 minutes at 14,000 rpm at 4°C. The supernatant was carefully removed and the pellet was air dried for 15 minutes and dissolved in 500 µl of double distilled water. The solution was applied to a microcon YM-30 centrifugal filter (Millipore, 42410) and spun for 12 minutes at 14,000×g at room temperature. This reduced the sample volume to ~10 µl. DNA was collected by inverting the microcon YM-30 centrifugal filter in a fresh tube and spinning for 3 minutes at 1,000×g at room temperature. The sample volume was adjusted to 12 µl with double distilled water. DNA concentration was measured using a NanoDrop spectrophotometer. Dam-methylated fragments were amplified as described previously (Vogel, M.J. et al. Detection of in vivo protein-DNA interactions using DamID in mammalian cells. Nat Protoc 2(6), 1467-78, 2007) using 500 ng of DNA as an input.
The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (QIAGEN, 28104) according to the manufacturer’s instructions. DNA was eluted in nuclease-free water (Ambion, AM9938). From 200 ng to 3 µg of the purified PCR products were diluted in 100 μl of nuclease-free water and transferred into a snap-cap microTUBE (6 × 16 mm) with AFA fiber (Covaris, 520045). DNA was sheared using the Covaris S2 instrument at the following settings: duty cycle = 10%, intensity = 5, cycles/burst = 200, time = 45 sec (as consequence, power = ~23W and bath temperature = ~4°C). The size of the DNA fragments before shearing was usually a smear in the range of 250–2000 bp with a peak at 600 bp, after shearing this was reduced to become a range of 100–500 bp with a peak around 300 bp. After shearing, the DNA was purified and concentrated using Agencourt magnetic beads (Beckman Coulter, A63881; 160 µl of beads were added to 100 µl of sheared DNA solution). DNA was eluted in 50 µl of nuclease-free water. DNA concentration was measured using an Agilent BioAnalyzer 2100 DNA 7500 chip (Agilent, 5067-1506). One μg of sheared material was used to prepare Illumina sequencing library with TruSeq DNA HT Sample Prep Kit (Illumina, FC-121-2003) according to the manufacturer’s instructions. Seven cycles of PCR were performed. This step included sample indexing. The library was quantified on an Agilent BioAnalyzer 2100 DNA 7500 chip that lists the product size distribution and quantifies the concentration in µg/µl and in nM.

treatment_protocol: Fat bodies were dissected from 10 larvae in 1×PBS [140 mM NaCl, 10 mM PO4 Buffer, 3 mM KCl; Invitrogen cat. no. 18912-014] using forceps and dissection needles and collected in 1×PBS solution on ice.

value: larval fat bodies

value: wandering third instar larvae

value: fat bodies

value: Dam4-HT-intein@L127C, induced by 25 µM 4-hydroxytamoxifen


title: FB.Pc.m_DamIDSeq

value: Dam-Polycomb


title: BR.HP1a.m_DamIDSeq

value: brain without ventral nerve cord


title: FB.HP1a.m_DamIDSeq