title: Fus

description:
RNA attached to Fus in Ezh2 wt cells
Protein crosslinked RNA
Processed data file: Fus.ensembl59.bed.gz

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 25 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 ug of α-Fus  antibody (Novus Biologicals #100-565)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

value: Fus Novus Biologicals #100-565


title: Suz12 Ezh2 d/d 1

status: Public on May 06 2016

type: SRA

city: London

first: Richard

last: Jenner

address: 72 Huntley Street

department: Cancer Biology

country: United Kingdom

institute: UCL Cancer Institute

description:
RNA attached to Suz12 in Ezh2 KO cells. Replicate1
Protein crosslinked RNA
Processed data file: Suz12_Ezh2dd.ensembl59.bed.gz

library_source: transcriptomic

data_processing:
Fastq files were demultiplexed and aligned using the iCount server
High-confidence peaks were identified using the 'peaks' option on the iCount server, with the parameters regionAsOne, flank 50, random perms 100 and fdr 0.05
Reads were filtered to remove any peaks within 50 nt of a repeatMasker-annotated repeat or a short non-coding RNA
Genome_build: mm9
Supplementary_files_format_and_content: BED files containing number of reads in each Ensembl59 gene for that iCLIP in the score column (column 5)

molecule: total RNA

organism: Mus musculus

source_name: mESC

growth_protocol: Cells were cultured on 0.1% gelatin-coated surfaces with KO-DMEM medium, 10% FCS, 5% knockout serum replacement, nonessential amino acids, L-glutamine, 2-mercaptoethanol, antibiotics (Invitrogen), and 1000 U/ml of leukaemia inhibitory factor (Abcam).

extract_protocol:
Individual-nucleotide resolution UV crosslinking and immunoprecipitation protocols were based in protocols available at Huppertz et al. Cells were irradiated with 0.15 J/cm2 of UV light in a Stratalinker 2400 (Stratagene) with bulbs emitting at 254nm, and 200 x10^6 cells per IP were lysed in 1ml of Lysis buffer (50 mM Tris–HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; plus protease inhibitors). Lysates were syringed with a 27G needle and incubated in a Thermomixer at 37°C and 1100 rpm for three minutes after addition of 200U/ml of DNase Turbo and of 20 U/ml of RNase I. IP was set up using 10 μl of α-SUZ12 (Cell Signaling #3737)  as described in iCLIP protocol. Beads were washed 3x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and Urea 1M), 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20) resuspended in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mM Tris-HCl, pH 6.5; 50m MgCl2 25mM dithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]) and incubated 30 min at 37°C 1100 rpm. Beads were washed 3x with 900 μl high-salt buffer, 2x with 900 μl wash buffer, resuspended in beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mM Tris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]), ligation was performed over night at 16°C and 1100 rpm. Beads were washed 3x with high-salt buffer, 2x wash buffer and 20% of the beads were incubated with 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water), radiolabelled and non-radiolabelled beads were pooled again and resuspended in 20 μl in Nupage loading buffer with reducing agent and run on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) in MOPS buffer according to the manufacturer's instructions. Protein-RNA complexes were transferred to a nitrocellulose membrane (Hybond, GE Healthcare) washed 2x with PBS1x and exposed to a Fuji film. Using the auto-radiograph as a mask RNA-protein complexes attached to the membrane were isolated and treated with 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) for 20 minutes at 1,100 rpm and 37°C. 200 μl of PK-urea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) was added and a second incubation was performed for 20 minutes at 37°C and 1100 rpm. Solution was collected and RNA was isolated by phenol: chloroform extraction and ethanol precipitation over night at -20°C.
RNA pellet was resuspended in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5 uM]; 0.5 μl dNTP mix [10mM]) and transferred in a 0.2 ml tube and incubated at 70°C. Retrotranscription was performed by addition of 2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen] and incubation 20 min at 42°C, 40 min at 50°C and 5 min at 80°C. cDNA was fractionated running samples on a precast 6% TBE-urea gel 180V 40 min and isolating bands at 250-180 nt (top), 120-180 nt (high), 85-120 nt (medium) and 70-85 nt (low). Gel slices were submerged and crushed in TE buffer, incubated 1h at 37°C 1100 rpm, flash frozen, and a second incubation was performed 1h at 37°C 1100 rpm. cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C. cDNA was resuspended in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl Circligase II [Epicentre]) transferred into a 0.2 tube and incubated for 1 h at 60°C for circularization. Samples were incubated 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]), oligo was annealed by temperature gradient and circular cDNA was re-linearized incubating samples with 2 μl BamHI (Fast Fermentas).
cDNA was isolated by phenol; chloroform extraction and ethanol precipitation over night at -20°C and pellet were resuspended in 20 μl of H20. Amplification was performed using 10 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen], 9 μl H20 and the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]n cycles, 68°C for 3 min, 4°C, with a number of cycles optimized previously. Library integrity was analysed using 8 μl PCR product with 8 μl of 5x TBE loading buffer and loading on a precast 6% TBE gel (Invitrogen). Gel was stained with Sybrgreen I (Invitrogen) and analyse with a gel imager. Library concentration was determined using the KAPA Universal Library Quantification kit, according to the manufacturer’s instructions and library concentration was corrected by multiplication by 0.38 to improve cluster density. Libraries were sequenced using Illumina HiSeq 2500 sequencers (50 bp single-end reads).

treatment_protocol: Ezh2fl/fl mESC were treated with 4-hydroxy-tamoxifen for 96 hours (800 nM, Sigma) when indicated to generate Ezh2∆/∆ cells

tag: strain

value: m ESC Ezh2 fl/fl

tag: treatment

value: Plus Tamoxifen

tag: genotype/variation

value: Ezh2 KO

tag: ip antibody

value: Suz12 Cell signalling #3737


title: Suz12 Ezh2 fl/fl 2

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate2
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz

value: No Tamoxifen

value: Ezh2 wild type


title: Suz12 Ezh2 d/d 3

description:
RNA attached to Suz12 in Ezh2 KO cells. Replicate3
Protein crosslinked RNA
Processed data file: Suz12_Ezh2dd.ensembl59.bed.gz


title: Suz12 Ezh2 fl/fl 1

description:
RNA attached to Suz12 in Ezh2 wt cells. Replicate1
Protein crosslinked RNA
Processed data file: Suz12_Ezh2flfl.ensembl59.bed.gz


title: Total_RNAseq_Ezh2_minusTx_1

description: Total RNAseq mESC wt cells Replicate1

data_processing:
Adaptor sequences were removed using fastq-mcf -t 0.0001 -l 15
Low quality bases were removed using seqtk trimfq -q 0.01
Reads were aligned to mm9 using tophat2 with the parameters --b2-very-sensitive -g 2 -p 4 --library-type fr-secondstrand
Reads in genes were quantified using bedtools intersect and normalised for gene length and GC content using EDAseq and DESeq2
Genome_build: mm9
Supplementary_files_format_and_content: tsv containing the normalised counts from DESeq2 for each gene

growth_protocol: Cells were cultured on  0.1% gelatin-coated surfaces with KO-DMEM medium, 10% FCS, 5% knockout serum replacement, nonessential amino acids, L-glutamine, 2-mercaptoethanol, antibiotics (Invitrogen), and 1000 U/ml of leukaemia inhibitory factor (Abcam).

extract_protocol:
Total RNA was purified from Ezh2fl/fl cells before and 4 days after treatment with tamoxifen (and Ezh2Δ/Δ cells) with TRizol and DNA removed with DNase-Turbo (Ambion)
Ribosomal RNA was depleted from total RNA with Ribo-Zero Gold (EpiCentre). RNA was fragmented using Tris pH 8 (40mM final), magnesium acetate (30mM final),potassium acetate (100mM final), and ends were repaired using Antartic phosphatase and T4 PNK (NEB) according manufacturer instruction.  Libraries were constructed using the Illumina Directional mRNA-Seq Sample Prep and the NEBNext Multiplex Small RNA Library Prep kits.


title: Total_RNAseq_Ezh2_plusTx_1

description: Total RNAseq mESC KO cells Replicate1


title: Total_RNAseq_Ezh2_minusTx_3

description: Total RNAseq mESC wt cells Replicate3


title: control_78h_rep3

city: Ann Arbor

first: Laura

last: Buttitta

state: MI

address: 5218 Biological Sciences Bldg., 1105 North University Ave.

department: Molecular, Cellular, Developmental Biology

country: USA

institute: University of Michigan

description: Found in "78h_ON" sheet of the processed file

data_processing:
Sequencing was performed with the Illumina HiSeq 2000 platform
High read quality was confirmed using FastQC
Reads were aligned to the D. melanogaster genome using Rsubread (v1.21.5) and featureCounts
Counts per million (cpm) were determined with edgeR (v3.13.4)
Transcripts with low expression were identified and removed using the data-based Jaccard similarity index determined with HTSFilter (v1.11.0)
The cpm were TMM normalized (calcNormFactors), voom transformed, then fit to a linear model (lmFit)
differential gene expression calls were made with eBayes
Genome_build: BDGP6.82
Supplementary_files_format_and_content: Raw counts of the sequencing reads per annotated gene were generated using Rsubread and featureCounts. The Microsoft Excel file contains counts for each biological replicate in separate columns. 48h and 78h of expression are located in the sheets labeled "48h_ON" and "78h_ON" respectively.

molecule: polyA RNA

organism: Drosophila melanogaster

source_name: wandering 3rd instar larval wing

growth_protocol: Animals were kept in un-crowded vials prior to their dissection

extract_protocol:
Twenty wing discs were dounce homogenized in TRIzol with ten strokes of a tight pestle. Samples were vortexed at speed 10 for 1min, incubated for 5mins at room temperature then frozen in liquid nitrogen. Samples were kept at -80°C until they were pooled to make a total of 40 wing discs per replicate of each condition and genotype. The pooled RNA was phase separated with chloroform and RNA was precipitated in isopropanol at -30°C overnight prior to 20min 4°C centrifugation at 12,000x g. The RNA pellet was resuspended in 30μL of RNase-Free water.
Using PolyA selection, the University of Michigan's Sequencing Core generated directional barcoded libraries for each sample and confirmed the quality via the Bioanalyzer and qPCR.

treatment_protocol: Animals were heat-shocked in a 37oC water bath for 30min. to induce overlapping clones. Transgene expression was silenced by keeping animals at 18°C until the animals were shifted to 28°C either 48h or 78h prior to their dissection as wandering L3 larvae

tag: tissue

value: wing

tag: developmental stage

value: third instar larva

tag: expression

value: control - 78 hours


title: control_78h_rep1


title: control_78h_rep2


title: Suz12 Jarid2 KO

description:
RNA attached to Suz12 in Jarid2 KO cells
Protein crosslinked RNA
Processed data file: Suz12_Jarid2KO.ensembl59.bed.gz

value: Jarid2 KO


title: Tip60dn_78h_rep3

value: dominant negative Tip60 - 78 hours


title: clr1D_Rep_1

city: Memphis

first: David

last: Finkelstein

state: TN

address: 332 N. Lauderdale St.

department: Computational Biology

institute: St Jude Children's Research Hospital

data_processing:
Illumina Casava1.8.2 software used for basecalling.
Reads were mapped to Schizosaccharomyces pombe using GSNAP (version 2013-02-25)
Gene level FPKM was estimated using in house perl script
Genome_build: ftp://ftp.ebi.ac.uk/pub/databases/pombase/pombe/Chromosome_Dumps/Schizosaccharomyces_pombe.ASM294v2.20.dna.genome.fa
Supplementary_files_format_and_content: tab-delimited text files include gene level FPKM valuse for each Sample

organism: Schizosaccharomyces pombe

source_name: Fission yeast

growth_protocol: Fission yeast (25 ml cultures) were grown to 2.5 x10-6 cells/ ml in YES media at 25C at 170 rpm

extract_protocol:
RNA was prepared as described (Creamer et al., MCB 34(11):2046-61, 2014)
Ribosomal RNA was removed using Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina, San Diego, CA.) Libraries were prepared from the rRNA depleted RNA using the TruSeq Stranded Total RNA Library Prep Kit with modifications (Brugger et al.)

tag: genotype

value: h- clr1D::KanMX4  ade6-210/216?  arg3D  his3D  leu1-32  ura4-D18


title: WT_Rep_3

value: PY 42


title: Tip60dn_78h_rep2


title: Tip60wt_78h_rep3

value: wild type Tip60 - 78 hours


title: Tip60wt_78h_rep2


title: Tip60wt_78h_rep1


title: Tip60dn_78h_rep1


title: control_48h_rep2

description: Found in "48h_ON" sheet of the processed file

value: control - 48 hours


title: control_48h_rep1


title: Tip60dn_48h_rep3

value: dominant negative Tip60 - 48 hours


title: Total_RNAseq_Ezh2_plusTx_3

description: Total RNAseq mESC KO cells Replicate3


title: preimmune_ChIPSeq

city: Moscow

first: Pavel

last: Georgiev

address: Vavilova 34/5

country: Russia

institute: Institute of Gene Biology

library_source: genomic

data_processing:
Read adapters were trimmed cutadapt with consequent windowed adaptive trimming using sickle (https://github.com/najoshi/sickle); reads shorter than 20 bps were discarded.
The remaining reads were mapped with Bowtie (the optimal alignments with "–best –strata –tryhard" parameters) to the bedgraphP r5 / UCSC dm3 Drosophila melanogaster genome assembly, and only uniquely mapped ("–m 1") reads were kept.
Reads overlapping with ENCODE blacklist regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were discarded.
Peak calling was done using MACS2 (2.1.0.20140616, https://github.com/taoliu/MACS) against preimmune and input control data.
Genome_build: UCSC dm3
Supplementary_files_format_and_content: Processed data was obtained by MACS and includes: bedGraph (bedgraph) read pileups and extended bed (narrowPeak) peak calls regions.

molecule: genomic DNA

source_name: Embryo

growth_protocol: Wild-type Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degrees. Embryos were collected for 12 hours. The collected embryos were dechorionated using 50% bleach and fixed in 10 ml cross-linking solution (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) with 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collection were set aside for staging.

extract_protocol:
Chromatin for input (0.5 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) was diluted in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted.   Sepharose was resuspended in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted.
To construct the libraries DNA was processed as described in the TruSeq RNA Sample Preparation Guide (Illumina) v2 from the end repair stage. This protocol has been chosen because of the very low amount of input DNA (<<10ng). Amplified libraries were quantified using fluorimetry with Qubit (Invitrogen, USA) and real-time PCR and diluted up to final concentration of 10 pM. Diluted libraries were clustered on a single read flowcell using cBot instrument and sequenced in 51 cycles using HiSeq2000 sequencer with TruSeq SBS Kit v3-HS (Illumina, USA). We sequenced an input sample as well as one sample for each of Pita, ZIPIC, Zw5 and preimmune chromatin immunoprecipitates.

treatment_protocol: Approximately 1g of Drosophila embryos were resuspended in 15ml ice-cold 0.1% Triton X-100 in PBS with protease inhibitors (PIs) and dounced 20x with loose pestle on ice, followed by centrifugation at 400g for 1min at 4 degrees. The supernatant was transferred to a fresh tube and centrifuged at 1100g for 10min at 4 degrees. The pellet was resuspended in 15ml ice-cold Cell lysis buffer with PIs (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP40) and dounced 20x with tight pestle. Two equal aliquots were transferred into 15ml falcon tubes and centrifuged at 2000g for 4min at 4 degrees. The pellets consisting of nuclei were resuspended in 1ml of ice-cold Nuclear Lysis Buffer with PIs (50 mM HEPES pH 8.0, 10 mM EDTA, 0.5% N-laurylsarcosine) and incubated for 20min at RT. 1ml ice-cold Nuclear Lysis Buffer with PIs was added to each tube and sonicated using the bioruptor. Chromatin was then transferred to 1.5 ml-eppendorf tubes and centrifuged at 14000 rpm for 10 min at 4 degrs. Finally, supernatants were pooled, aliquoted and frozen in liquid nitrogen. Chromatin (50 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) and incubated with anti-Pita, anti-ZIPIC, anti-Zw5 antibodies and rabbit IgG at 4 degrees on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 40 ul protein G sepharose at 4 degrees for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with 500 mM NaCl.

value: Oregon-R

value: Embryo

tag: chip antibody

value: preimmune rabbit sera


title: Tip60dn_48h_rep1


title: Total_RNAseq_Ezh2_plusTx_2

description: Total RNAseq mESC KO cells Replicate2

value: m ESC Ezh2 fl/fl

value: Plus Tamoxifen

value: Ezh2 KO


title: Tip60wt_48h_rep2

value: wild type Tip60 - 48 hours


title: Tip60wt_48h_rep1

value: wing


title: Total_RNAseq_Ezh2_minusTx_2

description: Total RNAseq mESC wt cells Replicate2

value: No Tamoxifen

value: Ezh2 wild type


title: Tip60wt_48h_rep3

value: third instar larva


title: Tip60dn_48h_rep2


title: SS020812 R1.1 co-culture

city: Richland

first: Lye Meng

last: Markillie

state: WA

address: 902 Battelle Blvd

department: Biosystems Dynamics & Simulation

institute: Environmental Molecular Sciences Laboratory

data_processing:
The 50-base short read sequences produced by the 5500XL SOLiD  sequencer were mapped in color space using SOLiD LifeScope software version 2.5 using the default parameters
Genome_build: Shewanella W3181 NC_008750
Genome_build: Synechococcus 7002 NC_010474, NC_010475, NC_010476, NC_010477, NC_010478, NC_010479 and NC_010480
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for both organism sample (*Syn7002.txt for Synechococcus and *SheW3181.txt for Shewanella)

organism:
Picosynechococcus sp. PCC 7002
Shewanella sp. W3-18-1

source_name: co-culture

growth_protocol: Shewanella W3-18-1 cultures were maintained at 30oC in tryptic soy broth or M1 defined medium amended with 20 mM D,L-Na lactate and 10 ml each of 10X Wolfe's vitamin and 10X mineral solutions (Kieft et al 1999). In chemostat cultures, Shewanella W3-18 was grown using 5 mM D,L-Na lactate as the sole source of C . Synechococcus 7002 was routinely grown in modified A+ media (Stevens and Porter 1980) at room temperature in 1-L Roux bottles sparged with 2% CO2-enriched air under continuous 50 µmolom-2os-1 illumination. Chemostat cultivation of Synechococcus 7002 was carried out using 8 mM NaHCO3 as the sole source of C in a white-light photobioreactor

extract_protocol:
RNA was extracted using Trizol following by DNAseI treatment using Qiagen RNAseay mini column
Template cDNA was prepared using the Applied Biosystems SOLiD® Total RNA-Seq Kit (Life Technologies, Carlsbad, CA) according to manufacturer's protocol. Sequencing was carried using the SOLiDTM 5500XL protocol (Life Technologies).

treatment_protocol: The co-cultivation experiments were carried out in a modified A+ medium which was contained 17 mM NH4Cl and 10 µm FeCl3·6H2O. D,L-Na lactate or NaHCO3 were used as the sole source of carbon at 5 mM and 8 mM concentrations

tag: shew l-limited

value: Shew W3-18 co-culture 8mM HCO3 light_limited


title: Zw5_ChIPSeq

value: Zw5 (rabbit, aa 98-302)


title: Plate2.Tunicamycin_H

status: Public on May 05 2016

city: Boston

first: Raivo

last: Kolde

state: MASSACHUSETTS

address: 185 Cambridge St.

department: Center for Computational and Integrative Biology

institute: Massachusetts General Hospital

description:
Agencourt AMPURE XP Purified Total RNA
12.5% lysate, 50ng Nextera

data_processing:
demultiplexing: The gene expression sequencing returns paired-end reads. Read 1 contains the plate-index, well-tag and RNA unique molecular identifier (UMI). Read 2 contains a fragment from the 3′ end of the transcript. The demultiplexing was done with a custom script that required perfect plate index and allowed one mismatch for the well tag.
mapping: The reads in read 2 were mapped to the refseq mouse mm10 gene using BWA with default parameters.
quantification: To quantify the expression genes we calculated the number of reads with unique UMI for each gene. The UMI information is embedded to the raw files as last 10 characters for every read description.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data file contains counts of unique RNA molecules for every gene.

source_name: BMDM

growth_protocol: BMDM cultured from wild-type BL6 mice at 8 weeks of age using conditioned media containing MCSF

extract_protocol:
RLT lysis, 50% SPRI purification, Turbo DNase, MaximaRT, SAv purification 25% (final 12.5% RNA lysate), Random hexamer 2nd strand cDNA, 50ng ds_cDNA tagmented for 12 minutes and run for 15 cycles PCR according to Nextera manufactures protocol
Modified SCRB-Seq, Soumillon, M., Cacchiarelli, D., Semrau, S., Oudenaarden, A. van & Mikkelsen, T. S. Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv 003236 (2014). doi:10.1101/003236; Modified Nextera protocol to PCR enrichonly  the 3' end of mRNA transcripts and create digital gene expression libraries for Illumina sequencing.

treatment_protocol: 30 minute pretreated with small molecules before 4 hour time point after LPS stimulation. RLT lysis in 100 uL and frozen at -80C.

value: bone marrow

tag: cell type

value: macrophage

tag: gender

value: male

tag: age

value: 8 weeks old

value: Tunicamycin

tag: stimulus

value: LPS 4h


title: Plate2.BrefA_H

value: BrefA


title: ZIPIC_ChIPSeq

value: Oregon-R

value: Embryo

value: ZIPIC (rabbit, aa 84-257)


title: Pita_ChIPSeq

value: Pita (rabbit, aa 550-683)


title: Plate2.ZVAD_H

value: ZVAD


title: Plate2.Caspase-1_H


title: Plate2.Halofuginone_H

value: Halofuginone


title: Plate2.Baflo_H

value: Baflo


title: Plate2.pGE2_H


title: control_48h_rep3

value: wing

value: third instar larva

value: control - 48 hours


title: Plate2.tBHQ_H

value: bone marrow

value: tBHQ


title: Plate2.DMSO_H

value: macrophage

value: male

value: DMSO


title: Plate2.Tunicamycin_G

value: 8 weeks old

value: Tunicamycin

value: LPS 4h


title: input_DNA

value: Oregon-R

value: Embryo


title: Plate2.BrefA_G

value: BrefA


title: Plate2.Halofuginone_G


title: Plate2.Caspase-1_G


title: Plate2.ZVAD_G

value: ZVAD


title: Plate2.Baflo_G


title: Plate2.pGE2_G


title: Plate2.Dexa_G

value: Dexa


title: Plate2.tBHQ_G


title: Plate2.Halofuginone_F

value: Halofuginone


title: Plate2.BrefA_F


title: Plate2.DMSO_G


title: Plate2.Tunicamycin_F


title: Plate2.Caspase-1_F


title: Plate2.Torin_H

value: Torin


title: Plate2.Torin_G


title: Plate2.Tunicamycin_E


title: Plate2.Baflo_F

value: Baflo


title: Plate2.pGE2_F


title: Plate2.Dexa_H


title: Plate2.Dexa_F


title: Plate2.tBHQ_F


title: Plate2.Torin_F


title: Plate2.BrefA_E


title: Plate2.Caspase-1_E


title: Plate2.Halofuginone_E


title: Plate2.Baflo_E


title: Plate2.ZVAD_E


title: Plate2.pGE2_E


title: Plate2.ZVAD_F


title: Plate2.Torin_E


title: Plate2.DMSO_E


title: Plate2.Dexa_E


title: Plate2.NoStim_D

value: NoStim


title: Plate2.NoStim_C

value: bone marrow


title: Plate1.Tunicamycin_H


title: Plate2.NoStim_A


title: Plate1.BrefA_H


title: Plate2.DMSO_F


title: Plate1.Halofuginone_H


title: Plate1.Caspase-1_H


title: Plate2.NoStim_B


title: Plate2.tBHQ_E

value: tBHQ


title: Plate1.pGE2_H

value: macrophage


title: Plate1.BrefA_G

value: male


title: Plate1.Baflo_H


title: Plate1.Dexa_H


title: Plate1.Halofuginone_G


title: Plate1.DMSO_H

value: DMSO


title: Plate1.ZVAD_G

value: 8 weeks old


title: Plate1.tBHQ_H


title: Plate1.Caspase-1_G


title: Plate1.ZVAD_H


title: Plate1.Torin_G


title: Plate1.Baflo_G


title: Plate1.Tunicamycin_G

value: Tunicamycin


title: Plate1.Dexa_G

value: LPS 4h


title: Plate1.Tunicamycin_F


title: Plate1.BrefA_F


title: Plate1.DMSO_G


title: Plate1.Halofuginone_F


title: Plate1.ZVAD_F


title: Plate1.pGE2_F


title: Plate1.Baflo_F


title: Plate1.tBHQ_F


title: Plate1.Caspase-1_F


title: Plate1.Torin_F


title: Plate1.Tunicamycin_E


title: Plate1.Halofuginone_E


title: Plate1.tBHQ_E


title: Plate1.Caspase-1_E


title: Plate1.pGE2_G


title: Plate1.pGE2_E


title: Plate1.Torin_E


title: Plate1.Torin_H


title: Plate1.Dexa_E


title: Plate1.DMSO_E


title: Plate1.Baflo_E


title: Plate1.tBHQ_G


title: Plate1.BrefA_E


title: Plate1.NoStim_D


title: Plate1.NoStim_C


title: Plate1.NoStim_B


title: KO2.HT.DNA.T4

description:
Agencourt AMPURE XP Purified Total RNA
3 ng total from 12.5% lysate

growth_protocol: BMDM cultured from WT and Myd88 KO bones shipped o/n on wet ice from Al Shaw lab by Jean Wilson at Yale, gown in 30% FBS DMEM +P/S and 20% CMG, replaced fresh 30% Serum Media and 20% CMG on Day4 when transducing in 10cm dishes with 10x 58-MPRA pathway virus. Cells grown in 10 cm petri dishes until day 9 when they were scraped and counted on the hemocytometer and replated in 96-well format at a density of 125K cells per well.

extract_protocol:
RLT lysis, 50% SPRI purification, Turbo DNase, MaximaRT, SAv purification 25% (final 12.5% RNA lysate), Random hexamer 2nd strand cDNA, 2ng ds_cDNA tagmented for 12 minutes and run for 15 cycles PCR according to Nextera manufactures protocol
Modified SCRB-Seq, Soumillon, M., Cacchiarelli, D., Semrau, S., Oudenaarden, A. van & Mikkelsen, T. S. Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv 003236 (2014). doi:10.1101/003236; Modified Nextera protocol to PCR enrichonly  the 3' end of mRNA transcripts and create digital gene expression libraries for Illumina sequencing.

treatment_protocol: 4 hour time course in 0.5hr intervals with 12 dependent and independent innate immune ligands. RLT lysis in 200 uL and frozen at -80C.

value: 13 weeks old

tag: plate

tag: myd88 genotype

value: KO

tag: replicate

value: HT-DNA

tag: time


title: KO2.TDB.T4

tag: strain

tag: treatment

value: TDB


title: KO2.Zymosan.T4

value: Zymosan


title: Plate1.DMSO_F


title: Plate1.Dexa_F

value: Dexa


title: KO2.MDP.LPS.T4

value: MDP/LPS


title: KO2.MDP.T4

value: MDP


title: KO2.CpG.T4

value: CpG


title: KO2.LPS.T4

value: bone marrow

value: LPS


title: KO2.R848.T4

value: macrophage


title: KO2.FLA.T4

value: male

value: FLA


title: KO2.Poly.I.C..T4


title: Plate1.ZVAD_E

value: 8 weeks old

value: ZVAD


title: KO2.HT.DNA.T3


title: KO2.TDB.T3


title: KO2.Pam3Cys.T4


title: KO2.Zymosan.T3


title: KO2.SeV.T3

value: SeV


title: KO2.MDP.T3

value: 13 weeks old


title: KO2.MDP.LPS.T3

value: KO


title: KO2.CpG.T3


title: KO2.LPS.T3


title: KO2.Pam3Cys.T3


title: KO2.Zymosan.T2.5


title: Plate1.NoStim_A

value: NoStim


title: KO2.SeV.T4


title: KO2.HT.DNA.T2.5

value: HT-DNA


title: KO2.R848.T3


title: KO2.TDB.T2.5

value: TDB


title: KO2.MDP.LPS.T2.5


title: KO2.SeV.T2.5


title: KO2.CpG.T2.5


title: KO2.Poly.I.C..T2.5


title: KO2.MDP.T2.5


title: KO2.R848.T2.5


title: KO2.Pam3Cys.T2.5


title: KO2.LPS.T2.5


title: KO2.Poly.I.C..T3


title: KO2.FLA.T3


title: KO2.SeV.T2


title: KO2.TDB.T2


title: KO2.MDP.LPS.T2


title: KO2.Zymosan.T2

value: Zymosan


title: KO2.MDP.T2


title: KO2.R848.T2


title: KO2.CpG.T2


title: KO2.LPS.T2


title: KO2.HT.DNA.T1.5


title: KO2.FLA.T2


title: KO2.Zymosan.T1.5


title: KO2.SeV.T1.5


title: KO2.Pam3Cys.T2


title: KO2.TDB.T1.5


title: KO2.Poly.I.C..T2


title: KO2.CpG.T1.5


title: KO2.R848.T1.5


title: KO2.LPS.T1.5


title: KO2.MDP.LPS.T1.5

value: MDP/LPS


title: KO2.MDP.T1.5

value: MDP


title: KO2.Pam3Cys.T1.5


title: KO2.SeV.T1


title: KO2.FLA.T2.5


title: KO2.HT.DNA.T2


title: KO2.MDP.LPS.T1


title: KO2.FLA.T1.5


title: KO2.Poly.I.C..T1.5


title: KO2.R848.T1


title: KO2.FLA.T1


title: KO2.Zymosan.T1


title: KO2.TDB.T1


title: KO2.LPS.T1


title: KO2.Poly.I.C..T1


title: KO2.MDP.T1


title: KO2.HT.DNA.T0.5


title: KO2.Pam3Cys.T1


title: KO2.FLA.T0.5


title: KO2.R848.T0.5


title: KO2.Zymosan.T0.5


title: KO2.LPS.T0.5


title: KO2.Zymosan.T0


title: KO2.SeV.T0.5


title: KO2.Poly.I.C..T0.5


title: KO2.CpG.T0

value: CpG


title: KO2.TDB.T0.5

value: bone marrow


title: KO2.MDP.T0.5


title: KO2.CpG.T0.5


title: KO2.HT.DNA.T1


title: KO2.Pam3Cys.T0.5


title: KO2.Poly.I.C..T0


title: KO2.MDP.LPS.T0.5


title: KO2.R848.T0


title: KO2.TDB.T0


title: KO2.MDP.LPS.T0


title: KO2.SeV.T0


title: KO2.HT.DNA.T0


title: KO2.MDP.T0


title: KO2.Pam3Cys.T0


title: KO2.FLA.T0


title: KO1.Zymosan.T4


title: KO1.LPS.T4

value: LPS


title: KO1.Pam3Cys.T4

value: macrophage


title: KO1.SeV.T4

value: male


title: KO1.MDP.LPS.T4


title: KO1.TDB.T4


title: KO1.Poly.I.C..T4


title: KO2.CpG.T1


title: KO1.MDP.T4


title: KO1.HT.DNA.T3


title: KO2.LPS.T0


title: KO1.R848.T4


title: KO1.SeV.T3


title: KO1.CpG.T4


title: KO1.R848.T3


title: KO1.TDB.T3


title: KO1.MDP.LPS.T3


title: KO1.Zymosan.T3


title: KO1.HT.DNA.T4


title: KO1.FLA.T3

value: FLA


title: KO1.HT.DNA.T2.5


title: KO1.LPS.T3


title: KO1.FLA.T4


title: KO1.TDB.T2.5


title: KO1.SeV.T2.5


title: KO1.Zymosan.T2.5


title: KO1.CpG.T3


title: KO1.MDP.LPS.T2.5


title: KO1.MDP.T2.5


title: KO1.Poly.I.C..T2.5


title: KO1.FLA.T2.5


title: KO1.CpG.T2.5


title: KO1.Poly.I.C..T3


title: KO1.Zymosan.T2


title: KO1.MDP.T3


title: KO1.SeV.T2


title: KO1.Pam3Cys.T2.5


title: KO1.R848.T2.5


title: KO1.Pam3Cys.T3


title: KO1.MDP.LPS.T2


title: KO1.MDP.T2


title: KO1.TDB.T2


title: KO1.LPS.T2


title: KO1.CpG.T2


title: KO1.R848.T2


title: KO1.Poly.I.C..T2


title: KO1.FLA.T2


title: KO1.Pam3Cys.T2


title: KO1.SeV.T1.5


title: KO1.HT.DNA.T1.5


title: KO1.TDB.T1.5


title: KO1.MDP.LPS.T1.5


title: KO1.MDP.T1.5


title: KO1.Zymosan.T1.5


title: KO1.HT.DNA.T2


title: KO1.Poly.I.C..T1.5


title: KO1.LPS.T1.5


title: KO1.FLA.T1.5


title: KO1.R848.T1.5


title: KO1.Pam3Cys.T1.5


title: KO1.CpG.T1.5


title: KO1.SeV.T1


title: KO1.R848.T1


title: KO1.MDP.T1


title: KO1.CpG.T1


title: KO1.TDB.T1


title: KO1.Zymosan.T1


title: KO1.LPS.T1


title: KO1.Zymosan.T0.5


title: KO1.HT.DNA.T1


title: KO1.Poly.I.C..T1


title: KO1.FLA.T1


title: KO1.Pam3Cys.T1


title: KO1.HT.DNA.T0.5


title: KO1.SeV.T0.5


title: KO1.MDP.LPS.T1


title: KO1.TDB.T0.5


title: KO1.MDP.LPS.T0.5


title: KO1.MDP.T0.5


title: KO1.FLA.T0.5


title: KO1.R848.T0.5


title: KO1.LPS.T0.5


title: KO1.CpG.T0.5


title: KO1.Poly.I.C..T0.5


title: KO1.Pam3Cys.T0.5


title: KO1.HT.DNA.T0


title: KO1.Zymosan.T0


title: KO1.TDB.T0


title: KO1.MDP.LPS.T0


title: KO1.MDP.T0


title: KO1.LPS.T0


title: KO1.SeV.T0


title: KO1.FLA.T0


title: KO1.Pam3Cys.T0


title: WT2.Zymosan.T4

value: WT


title: WT2.HT.DNA.T4


title: KO1.R848.T0


title: KO1.CpG.T0


title: WT2.MDP.T4


title: WT2.MDP.LPS.T4


title: WT2.R848.T4


title: WT2.CpG.T4


title: WT2.LPS.T4


title: WT2.Pam3Cys.T4


title: WT2.Poly.I.C..T4


title: WT2.HT.DNA.T3


title: WT2.Zymosan.T3


title: WT2.SeV.T3


title: WT2.CpG.T3


title: WT2.MDP.T3


title: WT2.TDB.T3


title: WT2.R848.T3


title: KO1.Poly.I.C..T0


title: WT2.MDP.LPS.T3


title: WT2.FLA.T3


title: WT2.Pam3Cys.T3


title: WT2.SeV.T4


title: WT2.LPS.T3


title: WT2.TDB.T4


title: WT2.Poly.I.C..T3


title: WT2.FLA.T4


title: WT2.SeV.T2.5


title: WT2.TDB.T2.5


title: WT2.MDP.T2.5


title: WT2.R848.T2.5


title: WT2.CpG.T2.5


title: WT2.LPS.T2.5


title: WT2.MDP.LPS.T2.5


title: WT2.FLA.T2.5


title: WT2.Poly.I.C..T2.5


title: WT2.HT.DNA.T2


title: WT2.Pam3Cys.T2.5


title: WT2.Zymosan.T2


title: WT2.SeV.T2


title: WT2.MDP.LPS.T2


title: WT2.CpG.T2


title: WT2.HT.DNA.T2.5


title: WT2.Zymosan.T2.5


title: WT2.R848.T2


title: WT2.MDP.T2


title: WT2.Poly.I.C..T2


title: WT2.HT.DNA.T1.5


title: WT2.Zymosan.T1.5


title: WT2.Pam3Cys.T2


title: WT2.FLA.T2


title: WT2.TDB.T1.5


title: WT2.SeV.T1.5


title: WT2.MDP.LPS.T1.5


title: WT2.FLA.T1.5


title: WT2.CpG.T1.5


title: WT2.R848.T1.5


title: WT2.Poly.I.C..T1.5


title: WT2.LPS.T1.5


title: WT2.MDP.T1.5


title: WT2.TDB.T2


title: WT2.Pam3Cys.T1.5


title: WT2.HT.DNA.T1


title: WT2.LPS.T2


title: WT2.TDB.T1


title: WT2.MDP.LPS.T1


title: WT2.MDP.T1


title: WT2.SeV.T1


title: WT2.Zymosan.T1


title: WT2.CpG.T1


title: WT2.R848.T1


title: WT2.FLA.T1


title: WT2.LPS.T1


title: WT2.Pam3Cys.T1


title: WT2.SeV.T0.5


title: WT2.Zymosan.T0.5


title: WT2.Poly.I.C..T1


title: WT2.R848.T0.5


title: WT2.LPS.T0.5


title: WT2.Poly.I.C..T0.5


title: WT2.TDB.T0.5


title: WT2.MDP.LPS.T0.5


title: WT2.CpG.T0.5


title: WT2.Pam3Cys.T0.5


title: WT2.FLA.T0.5


title: WT2.MDP.T0.5


title: WT2.TDB.T0


title: WT2.Zymosan.T0


title: WT2.SeV.T0


title: WT2.HT.DNA.T0


title: WT2.MDP.LPS.T0


title: WT2.FLA.T0


title: WT2.CpG.T0


title: WT2.LPS.T0


title: WT2.R848.T0


title: WT2.HT.DNA.T0.5


title: WT2.MDP.T0


title: WT2.Poly.I.C..T0


title: WT1.HT.DNA.T4


title: WT1.Zymosan.T4


title: WT1.SeV.T4


title: WT1.MDP.T4


title: WT1.MDP.LPS.T4


title: WT1.R848.T4


title: WT1.FLA.T4


title: WT1.Pam3Cys.T4


title: WT1.TDB.T3


title: WT1.MDP.LPS.T3


title: WT1.SeV.T3


title: WT1.HT.DNA.T3


title: WT1.MDP.T3


title: KO1.LPS.T2.5


title: WT1.R848.T3


title: WT1.LPS.T3


title: WT1.Poly.I.C..T3


title: WT1.Pam3Cys.T3


title: WT1.Zymosan.T2.5


title: WT1.HT.DNA.T2.5


title: WT1.CpG.T4


title: WT1.FLA.T3


title: WT1.SeV.T2.5


title: WT1.CpG.T3


title: WT1.TDB.T4


title: WT1.R848.T2.5


title: WT1.MDP.T2.5


title: WT1.MDP.LPS.T2.5


title: WT1.TDB.T2.5


title: WT1.FLA.T2.5


title: WT1.LPS.T4


title: WT1.CpG.T2.5


title: WT1.Poly.I.C..T4


title: WT1.LPS.T2.5


title: WT1.Poly.I.C..T2.5


title: WT1.Pam3Cys.T2.5


title: WT1.HT.DNA.T2


title: WT1.TDB.T2


title: WT1.MDP.LPS.T2


title: WT1.MDP.T2


title: WT1.R848.T2


title: WT1.LPS.T2


title: WT1.Poly.I.C..T2


title: WT1.Zymosan.T1.5


title: WT1.SeV.T1.5


title: WT1.MDP.T1.5


title: WT1.CpG.T1.5


title: WT1.R848.T1.5


title: WT1.MDP.LPS.T1.5


title: WT1.HT.DNA.T1.5


title: WT1.Pam3Cys.T1.5


title: WT1.HT.DNA.T1


title: WT1.TDB.T1.5


title: WT1.FLA.T1.5


title: WT1.LPS.T1.5


title: WT1.Poly.I.C..T1.5


title: WT1.SeV.T2


title: WT1.Zymosan.T2


title: WT1.Zymosan.T1


title: WT1.SeV.T1


title: WT1.TDB.T1


title: WT1.MDP.LPS.T1


title: WT1.FLA.T2


title: WT1.CpG.T2


title: WT1.Pam3Cys.T2


title: WT1.MDP.T1


title: WT1.CpG.T1


title: WT1.LPS.T1


title: WT1.Pam3Cys.T1


title: WT1.HT.DNA.T0.5


title: WT1.Poly.I.C..T1


title: WT1.Zymosan.T0.5


title: WT1.SeV.T0.5


title: WT1.MDP.LPS.T0.5


title: WT1.R848.T0.5


title: WT1.MDP.T0.5


title: WT1.TDB.T0.5


title: WT1.FLA.T0.5


title: WT1.CpG.T0.5


title: WT1.Pam3Cys.T0.5


title: WT1.LPS.T0.5


title: WT1.HT.DNA.T0


title: WT1.SeV.T0


title: WT1.Zymosan.T0


title: WT1.Poly.I.C..T0.5


title: WT1.MDP.T0


title: WT1.FLA.T0


title: WT1.TDB.T0


title: WT1.CpG.T0


title: WT1.R848.T1


title: WT1.FLA.T1


title: WT1.R848.T0


title: WT1.MDP.LPS.T0


title: T4.Control_11

description:
Agencourt AMPURE XP Purified Total RNA
12.5% lysate, 1ng NexteraXT

growth_protocol: BMDM cultured from WT  bones shipped o/n on wet ice from Luke O'Neil lab by Annie Curtis in Dublin Ireland, gown in 30% FBS DMEM +P/S and 20% CMG, replaced fresh 30% Serum Media and 20% CMG on Day2 and Day4 when transducing in 10cm dishes with 10x 58-TF-seq virus. Cells grown in 10 cm petri dishes until day 9 when they were scraped and counted on the hemocytometer and replated in 96-well format at a density of 125K cells per well.

extract_protocol:
RLT lysis, SPRI purification, Turbo DNase, MaximaRT, Random hexamer 2nd strand cDNA 50% of total first strand cDNA, 1ng of ds_cDNA per 96-well plate, 12 minutes and run for 16 cycles PCR according to Nextera manufactures protocol
Modified SCRB-Seq, Soumillon, M., Cacchiarelli, D., Semrau, S., Oudenaarden, A. van & Mikkelsen, T. S. Characterization of directed differentiation by high-throughput single-cell RNA-Seq. bioRxiv 003236 (2014). doi:10.1101/003236; Modified Nextera protocol to PCR enrich only  the 3' end of mRNA transcripts and create digital gene expression libraries for Illumina sequencing.

treatment_protocol: 4 hour dose curve on Halofuginone with and without LPS treatment

value: macrophages

value: 12 weeks old

tag: dose (nm)

value: Control


title: T4.Control_12

tag: tissue


title: WT1.LPS.T0

value: 13 weeks old


title: Smn Y107C transgene total-RNA-seq biol rep 1

city: Chapel Hill

first: Eric

middle: L

last: Garcia

state: NC

address: 3344 Genome Science Bldg

department: Lineberger Comprehensive Cancer Center

institute: University of North Carolina at Chapel Hill

description:
processed data file: gene_exp_kp_total.diff.gz
Y107C_1 / KP005_ACAGTG_L005

data_processing:
base-calling: CASAVA v.1.8.2
alignment: TopHat v2.0.8 with Bowtie v2.1.0 and Samtools v0.1.19
analysis of differential expression: Cufflinks v2.0.2
Genome_build: BDGP R5/dm3
Supplementary_files_format_and_content: Cufflinks differential expression (.diff) files for FPKMs. Median two-quartile wild-type normalized bedgraph files and non-normalized bedgraph files for inspecting mapped reads in genome browsers.

source_name: Smn Y107C transgene total-RNA-seq

growth_protocol: Larvae and pupae were cultured on molasses and agar at room temperature (24 ± 1°C). For pre-pupal collections, third instar larvae were transferred to vials.

extract_protocol:
Larvae were collected and washed with water at 74 ± 2 hours post egg laying. Larvae were then crushed in TRIzol and stored at -80°C. TRIzol extracted RNA was DNased with Ambion Turbo DNase.
Brown prepupae were collected and washed with water at < 12 hours post puparium formation. Prepupae were then crushed in TRIzol and stored at -80°C. TRIzol extracted RNA was DNased with Ambion Turbo DNase.
Illumina TruSeq RNA sample preparation kit v2

tag: genetic cross

value: SmnX7, SmnY107C/X7

value: < 12 hours post puparium formation

value: whole brown pre-pupae (n ≈ 30)


title: T4.Control_7


title: Smn V72G transgene total-RNA-seq biol rep 1

description:
processed data file: gene_exp_kp_total.diff.gz
V72G_1 / KP003_TTAGGC_L005

source_name: Smn V72G transgene total-RNA-seq

value: SmnX7, SmnV72G/X7


title: T4.Control_4


title: Smn wild-type transgene total-RNA-seq biol rep 2

description:
processed data file: gene_exp_kp_total.diff.gz
WT_2 / KP002_CGATGT_L005

source_name: Smn wild-type transgene total-RNA-seq

value: SmnX7, SmnWT/X7


title: Smn T205I transgene total-RNA-seq biol rep 2

description:
processed data file: gene_exp_kp_total.diff.gz
T205I_2 / KP008_ACTTGA_L005

source_name: Smn T205I transgene total-RNA-seq

value: SmnX7, SmnT205I/X7


title: WT1.Poly.I.C..T0


title: Smn wild-type transgene total-RNA-seq biol rep 1

description:
processed data file: gene_exp_kp_total.diff.gz
WT_1 / KP001_ATCACG_L005


title: WT1.Pam3Cys.T0


title: T4.Control_9


title: Smn T205I transgene total-RNA-seq biol rep 1

description:
processed data file: gene_exp_kp_total.diff.gz
T205I_1 / KP007_CAGATC_L005


title: Smn Y107C transgene total-RNA-seq biol rep 2

description:
processed data file: gene_exp_kp_total.diff.gz
Y107C_2 / KP006_GCCAAT_L005


title: T4.Control_10


title: Smn mutant total-RNA-seq biol rep 2

description:
processed data file: gene_exp_eg_total.diff.gz
Smn_2 / EG008_ACTTGA_L004

source_name: Smn mutant total-RNA-seq

value: 74 ± 2 hours post egg laying

value: whole larvae (n ≈ 30)


title: Smn V72G transgene total-RNA-seq biol rep 2

description:
processed data file: gene_exp_kp_total.diff.gz
V72G_2 / KP004_TGACCA_L005


title: T4.Control_6

value: bone marrow


title: Phax mutant total-RNA-seq biol rep 1

description:
processed data file: gene_exp_eg_total.diff.gz
Phax_1 / EG005_ACAGTG_L004

source_name: Phax mutant total-RNA-seq


title: T4.2.4nM_Halofuginone_2

value: Halofuginone


title: T4.Control_5

value: male


title: T4.Control_3


title: T4.Control_8


title: T4.Control_1


title: T4.9.7nM_Halofuginone_2


title: Smn mutant total-RNA-seq biol rep 1

description:
processed data file: gene_exp_eg_total.diff.gz
Smn_1 / EG007_CAGATC_L004


title: Phax mutant total-RNA-seq biol rep2

description:
processed data file: gene_exp_eg_total.diff.gz
Phax_2 / EG006_GCCAAT_L004


title: T4.9.7nM_Halofuginone_2mM_Proline_2


title: T4.2.4nM_Halofuginone_2mM_Proline_2


title: Phax mutant polyA-RNA-seq biol rep2

description:
processed data file: gene_exp_polyA.diff.gz
Phax_2 / EG006_CTTGTA_L005

source_name: Phax mutant polyA-RNA-seq


title: T4.2.4nM_Halofuginone_2mM_Proline_1


title: Phax mutant polyA-RNA-seq biol rep 1

description:
processed data file: gene_exp_polyA.diff.gz
Phax_1 / EG005_CAGATC_L005


title: T4.2.4nM_Halofuginone_1


title: Ars2 mutant polyA-RNA-seq biol rep 2

description:
processed data file: gene_exp_polyA.diff.gz
Ars2_2 / EG002_TGACCA_L005

source_name: Ars2 mutant polyA-RNA-seq


title: T4.39nM_Halofuginone_2mM_Proline_2


title: wild type total-RNA-seq biol rep 1

description:
processed data file: gene_exp_eg_total.diff.gz
Oregon R_1 / EG003_TTAGGC_L004

source_name: wild type total-RNA-seq

value: Oregon R


title: Ars2 mutant total-RNA-seq biol rep 1

description:
processed data file: gene_exp_eg_total.diff.gz
Ars2_1 / EG001_ATCACG_L004

source_name: Ars2 mutant total-RNA-seq


title: WT2.Pam3Cys.T0

description:
Agencourt AMPURE XP Purified Total RNA
3 ng total from 12.5% lysate

value: macrophage

value: WT


title: T4.Control_2

description:
Agencourt AMPURE XP Purified Total RNA
12.5% lysate, 1ng NexteraXT

value: macrophages

value: 12 weeks old


title: Ars2 mutant total-RNA-seq biol rep 2

description:
processed data file: gene_exp_eg_total.diff.gz
Ars2_2 / EG002_CGATGT_L004


title: DMOG_3

city: Bethesda

first: Yanqin

last: Yang

state: Maryland

address: 10 Center Dr.

institute: NIH

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then paired-end reads were mapped to the human genome (hg19, GRCh37) by Tophat 2.0.14 (bowtie v2.2.1.0, samtools v0.1.19.0) allowing 2 mismatches and 1 best hit
Mapped reads were counted in individual refSeq transcript region of each sample using RSeQC v2.6.1.  The R Bioconductor edgeR package was used to normalize raw reads and compare the difference.
Genome_build: hg19, GRCh37

organism: Homo sapiens

source_name: ND2 induced pluripotent stem cell-derived cardiomyocytes

growth_protocol: iPSCs were cultured in Essential 8 media and were subsequently differentiated based on a chemical modulation of Wnt-pathway protocol. Afterwards, cells were kept in cardiomyocyte maintainance medium.

extract_protocol:
RNA was harvested using RNAeasy Mini Kit from Qiagen. Illumina TruSeq Stranded Total RNA Library 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

treatment_protocol: iPSC-derived cardiomyocytes were treated on day 14 after initiation of the differentiation with either dimethyloxalylglycine (1 mM) or vehicle control for 18h.

value: day 15 iPSC-derived cardiomyocytes

value: wild-type


title: T4.156nM_Halofuginone_2


title: T4.39nM_Halofuginone_1


title: DMOG_2


title: DMOG_4


title: WT1.Zymosan.T3

value: 13 weeks old

value: Zymosan


title: DMOG_5


title: T4.625nM_Halofuginone_2


title: T4.625nM_Halofuginone_2mM_Proline_2


title: Ars2 mutant polyA-RNA-seq biol rep 1

description:
processed data file: gene_exp_polyA.diff.gz
Ars2_1 / EG001_CGATGT_L005


title: T4.9.7nM_Halofuginone_2mM_Proline_1


title: DMOG_1


title: T4.156nM_Halofuginone_1


title: wild-type total-RNA-seq biol rep 2

description:
processed data file: gene_exp_eg_total.diff.gz
Oregon R_2 / EG004_TGACCA_L004

source_name: wild-type total-RNA-seq


title: T4.2500nM_Halofuginone_2mM_Proline_2


title: T4.156nM_Halofuginone_2mM_Proline_2


title: Ctrl_4


title: T4.39nM_Halofuginone_2


title: Ctrl_5


title: T4.39nM_Halofuginone_2mM_Proline_1


title: Ctrl_2


title: T4.625nM_Halofuginone_2mM_Proline_1


title: T4.9.7nM_Halofuginone_1


title: Ctrl_3


title: T4.156nM_Halofuginone_2mM_Proline_1


title: T4.2500nM_Halofuginone_2mM_Proline_1


title: T4.2500nM_Halofuginone_2


title: 715R1 - shNHERF2

city: Atlanta

first: Viren

last: Patel

state: GA

address: 101 Woodruff Circle

department: EICC

institute: Emory University School of Medicine

description:
cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58674

data_processing:
Sequenced reads were mapped to the human reference genome UCSC version hg19 with TopHat version 1.3.3, and differentially expressed genes were assessed using Cuffdiff, a part of the Cufflinks version 1.3.0 package.
Please note that standard cufflinks/cuffdiff output files are provided in the compressed tar files as processed data and Cufflinks/Cuffdiff output file content/formats are described at:
https://cole-trapnell-lab.github.io/cufflinks/cuffdiff/#cuffdiff-output-files
https://cole-trapnell-lab.github.io/cufflinks/file_formats/
(also included in the 'readme.txt' file)
Genome_build: hg19
Supplementary_files_format_and_content: compressed tar files containing output of cufflinks/cuffdiff.

source_name: HCT116 xenograft tumors_shNHERF2

extract_protocol:
Qiagen miRNeAsy Mini Kit (Qiagen, cat # 217004)
Libraries were prepared according to the NEBNext RNA Seq protocol and sequenced on an Illumina 2500 HiSeq using v3 chemistry according to the Illumina Sequencing User’s Guide.  50M 100bp paired-end reads were generated per library (100M total reads per sample).  Base calling and fastq was done using Illumina’s HiSeq Control Software version 1.5.15.1 (RTA v1.13.18 and bclfastq v1.8.3).

value: HCT116 xenograft tumors

tag: genotype/variation

value: knockdown of NHERF2


title: 718R1 - shNHERF2

description:
cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58675


title: 714R1 - shNHERF2

description:
cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58673


title: T4.2500nM_Halofuginone_1


title: 721R1 - shLPA2

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58672

source_name: HCT116 xenograft tumors_shLPA2

value: knockdown of LPA2


title: Ctrl_1

tag: genotype


title: T4.10000nM_Halofuginone_2mM_Proline_2


title: 711R1 - shLPA2

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58671


title: 703R1 - pLK01

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz
SL58669

source_name: HCT116 xenograft tumors_pLKO


title: T4.10000nM_Halofuginone_2


title: 709R1 - shLPA2

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_shLPA2_vs_shNHERF2.tar.gz
SL58670


title: 701R1 - pLKO1

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz
SL58667


title: T1.Control_12

value: Control


title: T1.Control_11


title: T4.10000nM_Halofuginone_1


title: T1.Control_10


title: T1.Control_9


title: T1.Control_8


title: T4.10000nM_Halofuginone_2mM_Proline_1


title: ES87_shCuff_NRO

city: Pasadena

first: Maria

last: Ninova

state: CA

address: 1200 E California Blvd

department: Division of Biology and Biological Engineering

institute: Calfironia Institute of Technology

description:
BrdU-RNA
nuclear run-on sequencing

data_processing:
All RNA-sequencing datasets were first subjected to adaptor removal using the Reaper tool (v 12-205, Davis et al. 2013) or cutadapt tool (Martin, 2011) to eliminate adaptor sequences, and reads shorter than 15 nt were discarded
RNA-sequencing data was next filtered against Drosophila melanogaster  ribosomal RNA sequences using Bowtie 0.12.7 (Langmead et al., 2009), allowing up to 3 mismatches.
Pre-processed RNA-sequencing data, and ChIP-seq data, were mapped to D. melanogaster genome (UCSC dm3) retaining only uniquely mapping reads (-m 1) using Bowtie 0.12.7. Number of allowed mismatches (-v) were chosen depending on read lengths, with 0, 2 or 3 mismatches allowed for 50, 100 and 150 bp libraries, respectively. For small RNA libraries, 1 mismatch was allowed, and reads between 23-29 nt length were used for genome browser track generation.
For RNA-seq data, normalized coverage for 1 kb genomic window across the entire D. melanogaster genome was generated using custom scripts
genome build: UCSC dm3
processed data files format and content: Strand-specific genome coverage histograms for uniquely mapped reads in bigWig format for RNA-seq datasets (standard RNA, small RNA and Gro-seq data); and unstranded genome coverage histograms for uniquely maped reads in bigWig format for ChIP-seq datasets.
table of RPKM values for 1kb genomic windows

source_name: ovaries

extract_protocol:
Ovaries were dissected from adult flies and RNA was isolated with Ribozol Reagent. For standard non-polyA selected libraries, rRNA was depleted with Ribo-Zero™ rRNA Removal Kit. For polyA-selected libraries, selection was performed with the Dynabeads® mRNA Purification Kit. DNA for ChIP seq was prepared as in Le Thomas et al., 2014, using anti-RNA Pol II antibody from Abcam (ab5408). Nuclear run-on was performed as previously described (Shpiz et al.) with slight modifications. 5’-Bromouridine-5’-triphosphate (BrUTP; Sigma, B7166) labeled RNA was filtered through Illustra MicroSpin G25 columns (27-5325-01) twice to remove unincorporated BrUTP. The nuclear run-on RNA was captured using anti-BrdU antibody (Sigma, 032M 4753) by incubation for 1 hour followed by incubation with Protein G beads (Dynabeads, Invitrogen, 1003D) for 1 hour.
Standard RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit (NEB #E7420) according to the manufacturer's instructions. For global nuclear run-on (GRO-seq) (Core et al., 2008) immunoprecipitated RNA was used to clone total RNA libraries using the NEBNext Ultra Directional RNA Library Prep Kit (NEB #E7420). For small RNA librarires, 19-29 nt RNAs were isolated from total RNA of dissected ovaries using 12% polyacrylamide gels as described in previous studies (Aravin et al., 2008; Brennecke et al., 2007) and libraries were constructed using the Illumina TruSeq smallRNA Library Kit. ChIP-seq libraries were prepared using the NEBNext ChIP-Seq Library Prep Master Mix Set (#E6240).

value: 3-14 days old

tag: antibody


title: 702R1 - pLK01

description:
cuffdiff_out_pLKO1_vs_shLPA2.tar.gz, cuffdiff_out_pLKO1_vs_shNHERF2.tar.gz
SL58668


title: T1.Control_6

source_name: BMDM

value: bone marrow


title: T1.Control_7


title: ES89_control_NRO

description:
BrdU-RNA
nuclear run-on sequencing


title: ALT58_PldCRISPRmut

description:
Size-selected 19-29 nt RNA
small RNA sequencing

data_processing:
All RNA-sequencing datasets were first subjected to adaptor removal using the Reaper tool (v 12-205, Davis et al. 2013) or cutadapt tool (Martin, 2011) to eliminate adaptor sequences, and reads shorter than 15 nt were discarded
RNA-sequencing data was next filtered against Drosophila melanogaster  ribosomal RNA sequences using Bowtie 0.12.7 (Langmead et al., 2009), allowing up to 3 mismatches.
Pre-processed RNA-sequencing data, and ChIP-seq data, were mapped to D. melanogaster genome (UCSC dm3) retaining only uniquely mapping reads (-m 1) using Bowtie 0.12.7. Number of allowed mismatches (-v) were chosen depending on read lengths, with 0, 2 or 3 mismatches allowed for 50, 100 and 150 bp libraries, respectively. For small RNA libraries, 1 mismatch was allowed, and reads between 23-29 nt length were used for genome browser track generation.
Genome_build: UCSC dm3
Supplementary_files_format_and_content: Strand-specific genome coverage histograms for uniquely mapped reads in bigWig format for RNA-seq datasets (standard RNA, small RNA and Gro-seq data); and unstranded genome coverage histograms for uniquely maped reads in bigWig format for ChIP-seq datasets.


title: ES43_shWhite_tot_rRNAdep

description: rRNA depleted total RNA squencing chromatn libraries control


title: ES44_shCuff_tot_rRNAdep

description: rRNA depleted total RNA squencing chromatin libraries control


title: T1.Control_5


title: ALT57_PldCRISPRhet


title: T1.Control_3


title: KR08_nos-shCuff_polII_IP

description: ChIP

value: anti-RNA Pol II from Abcam (ab5408)


title: T1.Control_4

value: male


title: KR05_nos-shW_polII_IP

description: ChIP


title: KR01_nos-shWhite_input

description: input


title: KR02_nos-shCuff_input

description: input


title: T1.Control_2


title: T1.Control_1


title: T1.2.4nM_Halofuginone_2

value: Halofuginone


title: AC63_shWhite_polAsel

description: polyA seleted mRNA sequencing


title: T1.2.4nM_Halofuginone_2mM_Proline_2


title: T1.2.4nM_Halofuginone_1


title: AC64_shCuff_polAsel

description: polyA seleted mRNA sequencing


title: T1.9.7nM_Halofuginone_2mM_Proline_2


title: AC61_shWhite_rRNAdel

description: rRNA depleted total RNA squencing


title: AC62_shCuff_rRNAdel

description: rRNA depleted total RNA squencing


title: T1.9.7nM_Halofuginone_2


title: Brain_1B

city: Middlebury College

first: Amanda

last: Crocker

state: Vermont

address: 276 Bicentennial Way

department: Neuroscience

institute: Middlebury College

data_processing:
Multiplexing barcode: TCGAAG
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.

source_name: Brain

growth_protocol: Flies were raised at 25C.  Parents consisted of 5 females of the UAS-eGFP;UAS-eGFP;+ and 4 males of corresponding GAL4 line.  Parents were placed in a new vial every 4 days for 2 weeks to minimize overcrowding and keep the larval density relatively constant.  All flies were kept in a 12:12 Light Dark cycle.

extract_protocol:
Neurons were identified through anatomical location and GFP expression and then harvested into a patch pipet on an electrophysiology rig from alive flies.  Cells were harvested into .5ul Nuclease Free water to cause the cells to lyse.
cDNA was made and amplified using the Clontech SMARTer Ultra low RNA kit (version 2 HV). 15 rounds of amplification was perfromed on the samples.   This was then sheared to 200bp and put into an IXI Chip-seq protocol  (automated system) and barcoded following another 12 rounds of amplification. 12 samples were run in a lane and each sample was run across two lanes.

treatment_protocol: Animals undergoing memory formation were subjected to 8 spaced trainings of an odor (either 3-Octanol or 4-Methylcyclohexanol) with a 12 cycle 5 second 60V shock for 1 minute.

tag: cell class

value: UAS-GFP Background

tag: number cells

value: Millions

tag: training

tag: quality

value: more than >4 million mapped reads-single lane


title: T1.9.7nM_Halofuginone_2mM_Proline_1

tag: cell type

value: macrophages

tag: gender

tag: age

value: 12 weeks old


title: V3_WF_1B

description:
V3_WF_1
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: ACTTGA
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.

source_name: Whole Fly

value: CG0239-GAL4/UAS-GFP Background

value: more than >4 million mapped reads-across 2 lanes


title: T1.39nM_Halofuginone_2mM_Proline_2

description:
Agencourt AMPURE XP Purified Total RNA
12.5% lysate, 1ng NexteraXT

tag: replicate


title: Brain_1A

data_processing:
Multiplexing barcode: TACAGC
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: T1.9.7nM_Halofuginone_1

tag: time


title: V3_WF_2B

description:
V3_WF_2
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: GATCAG
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: V3_WF_4B

description:
V3_WF_4
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: TAGCTT
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: T1.39nM_Halofuginone_2

tag: strain


title: V3_WF_2


title: V3_WF_4


title: T1.39nM_Halofuginone_1

tag: treatment


title: V3_WF_1


title: T1.156nM_Halofuginone_2mM_Proline_2

tag: dose (nm)


title: T1.2.4nM_Halofuginone_2mM_Proline_1

tag: tissue


title: G386_WF_3B

description:
G386_WF_2A
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: GTGGCC
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.

value: G386-GAL4/UAS-GFP Background


title: T1.156nM_Halofuginone_2


title: T1.156nM_Halofuginone_2mM_Proline_1


title: G386_WF_4B

description:
G386_WF_2B
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: CGTACG
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: T1.156nM_Halofuginone_1


title: T1.625nM_Halofuginone_2


title: T1.625nM_Halofuginone_2mM_Proline_1


title: T1.625nM_Halofuginone_1


title: G386_WF_4


title: T1.2500nM_Halofuginone_2mM_Proline_2


title: T1.2500nM_Halofuginone_2


title: G386_WF_1

description:
G386_WF_1A
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: ATGTCA
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: c739_WF_2B

description:
c739_WF_2
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: ATCACG
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.

value: c739-GAL4/UAS-GFP Background


title: T1.2500nM_Halofuginone_1


title: c739_WF_2


title: c739_WF_4

description:
c739_WF_4
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: TTAGGC
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: T1.10000nM_Halofuginone_2mM_Proline_2


title: c739_WF_4B


title: c739_WF_1B

description:
c739_WF_1
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: TCGGCA
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: G386_WF_2

description:
G386_WF_1B
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: GTAGAG
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: c739_WF_1


title: T1.2500nM_Halofuginone_2mM_Proline_1

data_processing:
demultiplexing: The gene expression sequencing returns paired-end reads. Read 1 contains the plate-index, well-tag and RNA unique molecular identifier (UMI). Read 2 contains a fragment from the 3′ end of the transcript. The demultiplexing was done with a custom script that required perfect plate index and allowed one mismatch for the well tag.
mapping: The reads in read 2 were mapped to the refseq mouse mm10 gene using BWA with default parameters.
quantification: To quantify the expression genes we calculated the number of reads with unique UMI for each gene. The UMI information is embedded to the raw files as last 10 characters for every read description.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data file contains counts of unique RNA molecules for every gene.


title: T1.10000nM_Halofuginone_2mM_Proline_1


title: T1.625nM_Halofuginone_2mM_Proline_2


title: T1.10000nM_Halofuginone_1


title: G386_WF_1B


title: G386_WF_3


title: G386_WF_2B


title: NP1131_WF_3B

description:
NP1131_WF_2A
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: CTAGCT
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.

value: NP1131-GAL4/UAS-GFP Background


title: NP1131_WF_4B

description:
NP1131_WF_2B
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: CTCAGA
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.


title: NP1131_WF_1B

description:
NP1131_WF_1A
Samples run across two lanes and pooled

data_processing:
Multiplexing barcode: CACGAT
Cut and adapt the barcodes and remove poly A and T. Using Galaxy: Cutadapt: Removes sequences of 10 As or Ts along with barcodes.  Max error rate=.11, Matchtimes=1, Min Overlap length=9, Min length =36, Max length = 0,
Run Tophat2. Using Galaxy: Tophat2: Full parameter list: Max realign edit dist=1000, max edit dist =2, library type = FR Unstranded, Final read mismatches = 2, Use bowtie-n-mode = no, Anchor length = 8, max number mismatches in anchor region = 0, min length intron =70, max intron length = 500000, allow indel search = no, max number of alignments = 1, min intron length found during split-segment = 50, Max intron length found during split-segment = 500000, number of mismatches allowed in each segment alignment for reads mapped independently=2, Min length read segements= 25, Use own junctions = yes, Use Gene annotation model = Yes, Use raw junctions = no, Only look for supplied junctions = yes, Use coverage search= No, Use Microexon search = no, Do Fusion search = no, Set Bowtie2 settings = no, Specify read group = No
Run HTseqCounts. Using Galaxy: htseq-counts: Mode: Intersection Strict, Feature type: exon, ID Attribute: gene_id
DESEQ2. Using R: Deseq2 Version 1.4
Genome_build: ensembl_dmel_bdgp5.25; Drosophila_melanogaster.BDGP5.74.gtf
Supplementary_files_format_and_content: Htseq counts reported in .txt file.
Supplementary_files_format_and_content: A total of 4 files linked as supplementary files to the Series record:  One is an overall target file describing each sample in the combined data files and whether that sample was removed due to quality issues.  2nd file is the combined raw counts (HTseq-Count) for each sample we sequenced and set up so the rows are the genes and columns are the sample names.  3rd file is a file converting all gene counts to counts per million mapped reads (CPM). This file was used for z-scoring and removing genes with less than 2 CPM.  4th File is combined data normalized using DESeq2 and regularized log transformed, this file removed 5 samples (ab KC technicals, Brain samples and one G386 sample).  All genes for each cell type with <2CPM in half samples were replaced with zeros.