title: Shank2 splicing reporter, sorted cells with 2% highest EGFP/mCherry, replicate 1 (T15)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_12


title: Shank2 splicing reporter, unsorted cells, replicate 3 (T9)

status: Public on Oct 12 2018

type: SRA

city: Toronto

first: Ulrich

last: Braunschweig

state: Ontario

address: 160 College Street

department: Donnelly Centre

country: Canada

institute: University of Toronto

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_23

library_source: genomic

data_processing:
Library strategy: CRISPR screen
Base calls were processed with bcl2fastq2 (Illumina) version 2.18.
sgRNA sequences were extracted from raw reads based on matches to the tracrRNA and U6 promoter using custom Perl scripts, and perfect matches to expected sgRNAs were tallied.
To score sgRNAs that impact reporter fluorescence, raw counts from sorted and unsorted populations were analyzed with the count module of MAGeCK version 0.5.6 (Li et al., 2014) with default settings where 1,000 non-targeting control sgRNAs present in the Brie library were specified with parameter –control-sgrna.
Genome_build: Brie mouse sgRNA library (Addgene #73633)
Supplementary_files_format_and_content: Tab delimited files with counts per sgRNA sequence in each sample; MAGeCK.scores.xlsx is a speadsheet containing the results of MAGeCK analyses, i.e. the enrichment of genes in the sorted cell polulations.

molecule: genomic DNA

organism: Mus musculus

source_name: Neuroblastoma cells

growth_protocol: Mouse neuroblastoma (N2A) and 293T cells were grown in DMEM (high glucose; Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate, non-essential amino acids, and penicillin/streptomycin, at 37°C with 5% CO2.

extract_protocol:
cell engineering protocol: A Flp-in cassetted was integrated into the Rosa26 locus in N2a cells by first creating cells with an FRT site in that locus, followed by integration of a Flp-In construct from pcDNA5/FRT/TRE modified to include an rtTA3-compatible Tet Response Element (TRE). A bichromatic microexon splicing reporter based on a microexon from either murine Shank2 (ENSMUSE00001006087) or Mef2d (ENSMUSE00000673645) was then constructed by amplifying the microexons (extended by one nucleotide to cause a frame shift), flanking introns, upstream constitutive exon, and 20-50 nt of the downstream constitutive exon 5' of a fluorescence cassette consisting of EGFP and mCherry in different reading frames. This construct was cloned into a customized pcDNA5-based Gateway(R) compatible vector (Life Technologies) that contained 8x TET response elements followed by a miniCMV promoter, Kozak sequence, 3x Flag-tag, and attR sites, followed by integration in a clonal N2A Flp-In cell line.
Genomic DNA was extracted from the cell pellets of unsorted samples using the QIAamp Blood Maxi Kit (Qiagen) while gDNA from sorted populations was purified with the Midi Kit as per the manufacturer’s recommendations. Genomic DNA was precipitated using ethanol and sodium chloride, and resuspended in Buffer EB (10 mM Tris-HCl, pH 7.5).
gRNA inserts were amplified via PCR using primers harboring Illumina TruSeq adapters with i5 and i7 barcodes, following a two-step PCR approach.   In the first step, a total of 50 μg of gDNA was subjected to PCR (25 cycles, temperature of annealing (Ta) = 65°C) for enrichment of sgRNA cassettes using NEBNext Ultra II Q5 polymerase and staggered primers annealing to the end of the U6 promoter and the beginning of the tracrRNA. For the 2% highest or lowest expressing sorted cell populations 10 μg of gDNA was used. Subsequently, the individual PCR reactions were pooled and 50 μl of the first step PCR product was loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1.5 hour at 100V. PCR products of 200-230 bp were selected and the excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). 1/6th of the purified PCR products were subjected to 2nd step PCR (10 cycles, Ta= 62°C) using NEBNext Ultra II Q5 polymerase and primers harboring Illumina TruSeq adapters with i5 and i7 indices to generate barcoded sequencing libraries ready for Illumina sequencing (Table S8). The PCR amplicons were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100V. The libraries were size-selected at 270 bp and 300 bp, and the excised gel fragments were purified using Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen).  The purified libraries were analyzed for quality control, pooling and sequencing. Size confirmation analysis for each sample was performed on an Agilent Bioanalyzer dsDNA High Sensitivity chip. Amplicons were quantified using Kapa Universal qPCR Master Mix (Cat # KK4923, Roche) and Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and pooled at varied ratios by molarity after size-adjustment. To mitigate the effects of index hopping, pooling was performed just prior to sequencing. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (Cat # E7630L, NEB).

treatment_protocol: 80 million N2A Flp-In cells expressing bichromatic splicing reporters were infected with the lentiviral Brie library (78,637 gRNAs) at an MOI ~0.3, in the presence of 8 μg/ml polybrene, such that every sgRNA was represented in approximately 300 cells. 24 hours after infection, the medium was replaced with fresh medium containing Puromycin (2.5 μg/ml) and cells were incubated for an additional 72 hours. After selection cells were split such that each sgRNA would be represented by an average of 300 cells in the population (i.e. 24 million cells) and passaged every three days.   For cell sorting, the 24 million cells were seeded and the next day 1 μg/ml doxycycline was added to induce bichromatic reporter expression. 24 hours later the cells were harvested and resuspended in sorting buffer (Hanks Balanced Salt Solution, 25 mM HEPES pH 7.0, 1 mM EDTA, 1% Albumin) at a concentration of 5 million cells per ml. The cells were passed through a nylon mesh with a pore size of 40 μm to eliminate large aggregates. Filtered cells were sorted based on the relative EGFP:mCherry expression FACS. For each replicate of the lowest/highest 2% sorting scheme, 300,000-500,000 cells were collected. For the 30% sorting scheme, the same procedure was followed except that the 30% of cells with the highest or lowest EGFP:mCherry ratio were sorted. In addition, 24 million cells were also collected prior to sorting as a reference point for comparing enrichment in the sorted populations.

tag: cell line

tag: cell type

value: Neuroblastoma


title: Shank2 splicing reporter, unsorted cells, replicate 2 (T17)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_13


title: Shank2 splicing reporter, unsorted cells, replicate 5 (T24)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_31


title: Shank2 splicing reporter, unsorted cells, replicate 4 (T22)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_27


title: Shank2 splicing reporter, unsorted cells after infection (T0)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_3


title: A549 cells infected by Influenza virus PR8 replicate 3

city: Stillwater

first: CHAOQUN

last: HUANG

state: OK

address: 264 McElroy Hall

department: Physiological Sciences

country: USA

institute: Oklahoma State University

library_source: transcriptomic

data_processing:
The indexed libraries were pooled in equal molar ratio and sequenced on the Illumina NextSeq 500 system using 2x75bp paired-end sequencing.
Paired-end reads were directionally mapped to the genomic loci of lncRNA (GRCh37 /hg19) by TopHat2.
Cufflinks/Cuffdiff analysis was run to identify the dysregulated RNAs.
Genome_build: GRCh37 /hg19
Supplementary_files_format_and_content: *.fpkm_tracking: Tab-delimited text files include FPKM values generated by Cufflinks.

molecule: total RNA

organism: Homo sapiens

source_name: A549 cells

extract_protocol:
Total RNA was isolated from A549 cells by using Tri Reagents.
Libraries were prepared with the Illumina TruSeq Stranded mRNA Library Preparation kit. Briefly, poly-adenylated mRNA were selected using oligo-dT beads and fragmented to 300-500bp. Each fragment undergoes cDNA synthesis using random hexamer. Indexed adapters were ligated to the resulting cDNA.

value: Adenocarcinomic lung epithelial cells

tag: gender

value: male

tag: treatment

value: Influenza virus PR8 infection


title: Shank2 splicing reporter, unsorted cells, replicate 1 (T15)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_9


title: A549 cells infected by Influenza virus PR8 replicate 2


title: A549 cells infected by Influenza virus PR8 replicate 1


title: A549 cells treated with PBS replicate 3

value: Mock infection


title: A549 cells treated with PBS replicate 1


title: A549 cells treated with PBS replicate 2


title: Shank2 splicing reporter, sorted cells with 2% highest EGFP/mCherry, replicate 4 (T22)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_29


title: Shank2 splicing reporter, sorted cells with 2% highest EGFP/mCherry, replicate 2 (T17)

description:
Counts_Shank2.tab
MAGeCK.scores.xlsx
sample name in processed data file: BB_16


title: WiDr-vemurafenib treated

city: Oakland

first: Ravi

last: Sachidanandam

state: CA

address: 2945 Webster St

institute: Girihlet Inc.

data_processing:
Reads were split into two 37 mers and mapped exactly to the human genome (hg38). Using only reads that mapped once to the genome, and the underlying gene annotations, reads were assigned to genes. The counts were then quantile normalized across the set. to create the table shown in the processed data file, which was used to draw conclusions.
Genome_build: hg38
Supplementary_files_format_and_content: quantile_normalized_processed.csv

source_name: Cancer cell line

growth_protocol: Cancer cell lines were maintained in a humidified incubator at 37 °C with 5% CO2, cultured in RPMI 1640 supplemented with 10% FBS, 2mM glutamine and 100 IUml−1 penicillin/streptomycin.

extract_protocol:
Total RNA was isolated using TRIzol (Invitrogen) from DESCRIBE SAMPLE.
polyA-tailed mRNA was selected using beads with oligo-dT, fragmented, cDNAs were created using random-hexamers and ligated with bar-coded adaptors compatible with NextSeq500.

treatment_protocol: Cells were treated with vemurafenib (2µM) for 24 hours

tag: tissue

value: Colon

tag: disease

value: colorectal adenocarcinoma

tag: genotype

value: BRAF (V600E)


title: Hth104-Control

value: Thyroid

value: Thyroid gland undifferentiated (anaplastic) carcinoma


title: SW1736-vemurafenib treated


title: Day_250_3

city: Baltimore

first: Robert

middle: J

last: Johnston

state: MD

address: 3400 N Charles St, Mudd Hall Room 314

department: Biology

institute: Johns Hopkins University

data_processing:
Base calls performed using RTA v2
Expression levels were quantified using Kallisto (version 0.34.1) with the following parameters: "-b 100 -l 200 -s 10 -t 20 --single".
The Gencode release 28 comprehensive annotation was used as the reference transcriptome
Genome_build: Gencode release 28
Supplementary_files_format_and_content: Excel sheet with average TPM values for each timepoint are included, with coresponding standard deviation in the second tab

molecule: polyA RNA

source_name: Retina

growth_protocol: Retinas were differentiated from stem cells as described in Eldred et al. 2018, “Thyroid hormone signaling specifies cone subtypes in human retinal organoids”.

extract_protocol:
RNA from individual organoids was extracted using the Zymo Direct-zol RNA Microprep Kit (Zymo Research) according to manufacturer’s instructions.
Individual libraries were prepared for each sample using the Illumina TruSeq stranded mRNA kit ; Libraries were sequenced on an Illumina NextSeq 500 with single 200 bp reads

value: iPSC derived Retinal Tissue

tag: cell background

value: IMR-90 human fetal lung fibroblast cells

tag: differentiation day


title: SW1736-Control


title: Day_250_1


title: Day_250_2


title: Day_200_2


title: Day_200_3


title: WiDr-Control


title: Hth104-vemurafenib treated


title: Day_181_1


title: Day_181_3


title: Day_181_2


title: Day_200_1


title: Day_173_3


title: Day_158_2


title: Day_173_2


title: Day_173_1


title: Day_111_3


title: Day_128_3


title: Day_128_1


title: Day_128_2


title: Day_158_1


title: Day_111_1


title: Day_69_3


title: Day_111_2


title: Day_69_2


title: Day_69_1


title: Day_35_3


title: Day_35_2


title: Day_35_1


title: Day_10_1


title: Day_10_3


title: human_atac_pk_basket

city: New York

first: Xiao

last: Xu

state: NY

address: 1230 York Ave

department: Laboratory of Molecular Biology

institute: The Rockefeller University

data_processing:
Adapter sequences were trimmed and reads were filtered depending on quality using Trim Galore! (trim_galore --stringency 3 --fastqc --paired *_R1_2.fastq.gz *_R2_2.fastq.gz).
Reads that passed the filter were aligned using Bowtie2 (Langmead and Salzberg, 2012) with parameters “-X 2000 --no-mixed --no-discordant” to the Homo sapiens reference genome (hg38).
Duplicate reads were removed using SAMtools (Li et al., 2009).
Reads were normalized to 1x depth (reads per genome coverage, RPGC) and input using deepTools (Ramirez et al., 2016) bamCompare module, ignoring chrX, chrY, chrM and filtering reads for minimum mapping quality of 30.
For data visualization, tdf files were generated using igvtools.
Unique fragments under 100 nt were selected as they could not contain a nucleosome. These fragments were used to call peaks with MACS2 with parameters “--nomodel -q 0.01 --call-summits --keep-dup all”. The summits for each peak from the replicates were extended to 500bp using bedtools slop. The peaks were filtered to remove chrY and chrM peaks and peaks that overlap with the hg38 blacklist from ENCODE (Consortium, 2012; Kundaje, 2013).
Genome_build: hg38
Supplementary_files_format_and_content: tdf format for visualization in IGV
Supplementary_files_format_and_content: txt format of called peaks

source_name: ATAC-seq using nuclei from specific cell types

extract_protocol:
Nuclei were isolated from post-mortem cerebellar tissue. ATAC-seq libraries were prepared as described previously (Buenrostro et al., 2013; 2015) with the following modifications: nuclei were resuspended in ice-cold lysis buffer, omitting incubation step on ice and centrifuged. Transposition reaction was performed for 30 min at 37°C using Tn5 transposase from Nextera. DNA fragments were purified and size selected using AMPure XP beads to yield 100-700bp fragments.
Libraries were generated using the Nextera DNA Library Preparation Kit. ATAC-seq libraries were sequenced on Illumina NextSeq 500 to obtain 75bp paired-ended reads.

value: female

tag: age

tag: brain region

value: cerebellum


title: RNA-Seq of N2A cells after knockdown of Rbfox2, replicate 1

description:
vast-tools.AltSplicing_Srrm3.Srrm4_Srsf11_Rbfox2.tab
AltSplicing_Srrm3.Srrm4_Srsf11_Rbfox2.tab
Expression_Srrm3.Srrm4_Srsf11_Rbfox2.tab

data_processing:
Base calls were processed with bcl2fastq2 (Illumina) version 2.18.
RNA-seq reads were processed for alternative splicing and expression analysis using vast-tools, version 1.0 (Tapial et al., (2017)), which is available on github (https://github.com/vastgroup/vast-tools).
Alternative splicing analysis: From primary vast-tools output, events with poor coverage or junction balance were filtered out (vast-tools quality column score 3 other than SOK/OK/LOW for cassette exon [CE], microexon [MIC] and alternative 5'/3'-ss events or coverage less than 15 reads for intron retention [IR] events; score 4 other than OK/B1 for CE and MIC events or less than 0.05 for IR events). Significantly differential AS was assessed with the diff module of vast-tools under development and were additionally required to have a PSI difference >10.
Expression analysis: Expression differences were calculated based on vast-tools raw read counts per gene using the R package edgeR.
Genome_build: mm9
Supplementary_files_format_and_content: All files are in tab-delimited format. Files vast-tools.AltSplicing_Rnps1.tab and vast-tools.AltSplicing_Srrm3.Srrm4_Srsf11_Rbfox2.tab contain primary vast-tools output. Files AltSplicing_Rnps1.tab and AltSplicing_Srrm3.Srrm4_Srsf11_Rbfox2.tab contain filtered PSI values and annotations of differential AS based. Files Expression_Rnps1.tab and Expression_Srrm3.Srrm4_Srsf11_Rbfox2.tab each contain raw counts per gene as well as fold changes and associated FDR.

growth_protocol: Mouse neuroblastoma (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin, and maintained at 37°C with 5% CO2.

extract_protocol:
Total RNA was extracted with the Qiagen RNasy Mini kit.
1000 ng per sample was used in the library preparation. To obtain larger insert size (540 nt on average after size selection), Illumina’s TruSeq Stranded mRNA sample preparation protocol was modified as follows: 1) fragmentation time and temperature was reduced from 8 minutes at 98 C to 2 minutes at 80 C. Average size distribution prior to library preparation was 232 nt versus 740 nt, respectively; 2) after the final library was generated, the samples were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100 V. The libraries were size selected between 400 and 600 bp. Excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). The rest of the protocol was adapted as described in Illumina’s TruSeq Stranded mRNA sample preparation guide (Part# 15031047 Rev. E, Illumina).

treatment_protocol: Cells were transfected with siGENOME siRNA smartpools (Dharmacon) using Lipofectamine RNAiMAX (Invitrogen), as recommended by the manufacturer. A non-targeting siRNA pool was used as a control. Cells were harvested 48 hours post transfection.

tag: knockdown


title: RNA-Seq of N2A cells after knockdown of Rbfox2, replicate 2


title: Day_20_1


title: human_atac_xk_granule


title: Day_10_2


title: SST4: SST Control4

city: New York City

first: Alireza

last: Khodadadi-Jamayran

address: 550 1st Ave, MSB 304

department: Division of Advanced Research Technologies (DART)

institute: New York University, NYU Langone Medical Center

data_processing:
Basecalls performed using CASAVA
All the reads were mapped to mm10 using STAR guided by GTF file
Calculating insert size and standard deviation performed using Picard tools
cufflinks and cuffdiff were used for transcript assembly and differential expression analysis
rMATS was used for event-based alternative splicing analysis
BAM to bedgraph performed using bedtools
bedgraph to bigwig performed using bedGraphToBigWig v4
Statistics and plotting performed in R v3.3.0
Genome_build: mm10
Supplementary_files_format_and_content: BigWig (coverage for mapped reads)

source_name: Brain slices

extract_protocol: Brain slices were treated with pronase, S1 cortex was dissected, dissociated for FACS of SST+ or PV+ neurons at P8

value: S1 cortex

tag: tag

value: SST+ cortical interneuronals


title: SST1: SST Control1


title: PV-M7: PV Mutant7

value: PV+ cortical interneuronals


title: SST3: SST Control3


title: Day_20_2


title: PV-M3: PV Mutant3


title: PV-M2: PV Mutant2


title: PV-M1: PV Mutant1


title: PV-M8: PV Mutant8


title: PV-M5: PV Mutant5


title: PV-C3: PV Control3


title: PV-C4: PV Control4


title: Brie library plasmid prep

description:
Counts_Shank2.tab
sample name in processed data file: BB_1

organism: synthetic construct

source_name: Isolated plasmid DNA

extract_protocol:
Plasmid preparation used to generate lentivirus
Genomic DNA was extracted from the cell pellets of unsorted samples using the QIAamp Blood Maxi Kit (Qiagen) while gDNA from sorted populations was purified with the Midi Kit as per the manufacturer’s recommendations. Genomic DNA was precipitated using ethanol and sodium chloride, and resuspended in Buffer EB (10 mM Tris-HCl, pH 7.5).
gRNA inserts were amplified via PCR using primers harboring Illumina TruSeq adapters with i5 and i7 barcodes, following a two-step PCR approach.   In the first step, a total of 50 μg of gDNA was subjected to PCR (25 cycles, temperature of annealing (Ta) = 65°C) for enrichment of sgRNA cassettes using NEBNext Ultra II Q5 polymerase and staggered primers annealing to the end of the U6 promoter and the beginning of the tracrRNA. For the 2% highest or lowest expressing sorted cell populations 10 μg of gDNA was used. Subsequently, the individual PCR reactions were pooled and 50 μl of the first step PCR product was loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1.5 hour at 100V. PCR products of 200-230 bp were selected and the excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). 1/6th of the purified PCR products were subjected to 2nd step PCR (10 cycles, Ta= 62°C) using NEBNext Ultra II Q5 polymerase and primers harboring Illumina TruSeq adapters with i5 and i7 indices to generate barcoded sequencing libraries ready for Illumina sequencing (Table S8). The PCR amplicons were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100V. The libraries were size-selected at 270 bp and 300 bp, and the excised gel fragments were purified using Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen).  The purified libraries were analyzed for quality control, pooling and sequencing. Size confirmation analysis for each sample was performed on an Agilent Bioanalyzer dsDNA High Sensitivity chip. Amplicons were quantified using Kapa Universal qPCR Master Mix (Cat # KK4923, Roche) and Qubit dsDNA HS Assay Kit (Cat # Q32854, Thermo Fisher Scientific) and pooled at varied ratios by molarity after size-adjustment. To mitigate the effects of index hopping, pooling was performed just prior to sequencing. The final pool was run on an Agilent Bioanalyzer dsDNA High Sensitivity chip and quantified using NEBNext Library Quant Kit for Illumina (Cat # E7630L, NEB).


title: PV-C1: PV Control1


title: ARPE19_VitC_2_IN

city: Provo

first: David

middle: Wayne

last: Sant

state: UT

address: 1712 South East Bay Blvd, Building 5 Suite 300

department: Biomedical Informatics

institute: Noorda College of Osteopathic Medic

description:
Input DNA
Counts of reads within each peak investigated

data_processing:
Basecalls performed using CASAVA version 1.4
Bases were quality trimmed to q20 and adaptors were removed using Trim_Galore!
RNA-seq reads were aligned to the HG38 transcriptome using STAR
RNA-seq counts were generated during alignment using STAR
hMeDIP-seq reads and Unprecipitated Input reads were aligned to the HG38 genome assembly using BWA
Peak calling was performed with MACS2
Peaks were filtered using the IDR pipeline from the Encode Project with an IDR cutoff of 0.01.
Number of reads in peaks were counted using HTSeq-count.
Genome_build: HG38
Supplementary_files_format_and_content: Count files were generated using either STAR or HTSeq-count.

source_name: Retinal Pigment Epithelial Cells

extract_protocol:
Qiagen RNEasy or Qiagen DNA Mini
Active Motif hMeDIP or Epicentre RiboZero

treatment_protocol: 50 micromolar sodium ascorbate (Vitamin C)

value: ARPE-19 Cells

value: 50 micromolar Vitamin C 7 Days


title: ARPE19_VitC_1_IN


title: Villin-knockdown HepG2 cells

type: RNA

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: 10-1 tebiro 6-chome

country: Japan

institute: Toray Industries,Inc.

description: Total RNA was prepared at 72 hrs.

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

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

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

source_name: HepG2 cells

label_protocol: cDNA and Amino Allyl aRNA was synthesis by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyeDye Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc..

growth_protocol: HepG2 cells were cultured in minimum essential medium supplementedwith 10% Fetal bovine serum  and antibiotics/antimycotics in a 5% CO2 incubator at 37˚C .

extract_protocol: Total RNA was prepared using TRIzol (Life Technologies) according to the manufacture's method followed by RQ1 Rnase-free DNase treatment (Promega).

treatment_protocol: Silencer Select siRNA oligonucleotides targeting villin (Life Technologies; s14792, s14797) or PTK7 (s11490) as well as negative control siRNA were transfected using RNAiMAX (Life Technologies) at the final concentration of 10 nM and cultured for 72 hrs.

tag: sirna

value: Villin knockdown


title: Negative control

description: Total RNA was prepared at 72 hrs.

value: Negative control


title: M4: SST Mutant4


title: ARPE19_Control_2_IN

value: Control


title: ARPE19_Control_1_IN


title: M3: SST Mutant3


title: M2: SST Mutant2


title: PV-M6: PV Mutant6


title: PTK7-knockdown HepG2 cells

description: Total RNA was prepared at 72 hrs.

value: PTK7 knockdown


title: ARPE19_VitC_1_IP

description:
Anti-5hmC Immunoprecipitated
Counts of reads within each peak investigated


title: ARPE19_VitC_2_IP


title: C5: SST Control 5


title: ARPE19_VitC_3_RNA

description:
Ribodepleted RNA
Counts of reads in each gene


title: ARPE19_Control_3_RNA


title: M1: SST Mutant1


title: C1: SST Control 1


title: C3: SST Control 3


title: ARPE19_Control_2_IP


title: C2: SST Control 2


title: ARPE19_VitC_1_RNA


title: C4: SST Control 4


title: RNA-Seq of N2A cells after non-targeting knockdown (control for Srrm3/Srrm4, Srsf11 and Rbfox2 knockdowns), replicate 2

value: Neuroblastoma

value: siNT


title: RNA-Seq of N2A cells after knockdown of Srsf11, replicate 2


title: ARPE19_Control_1_RNA


title: RNA-Seq of N2A cells after knockdown of Srrm3 and Srrm4, replicate 2


title: RNA-Seq of N2A cells after knockdown of Srsf11, replicate 1


title: RNA-Seq of N2A cells after knockdown of Srrm3 and Srrm4, replicate 1


title: M5: SST Mutant5


title: RNA-Seq of N2A cells after non-targeting knockdown (control for Srrm3/Srrm4, Srsf11 and Rbfox2 knockdowns), replicate 1


title: RNA-Seq of N2A cells after non-targeting knockdown (control for Rnps1 knockdown), replicate 2

description:
vast-tools.AltSplicing_Rnps1.tab
AltSplicing_Rnps1.tab
Expression_Rnps1.tab


title: RNA-Seq of N2A cells after knockdown of Rnps1, replicate 1


title: iCLIP-seq of Flag-EGFP in differentiated neurons, replicate 1

description:
Extracted RNA was reverse transcribed using primer Rt14clip: /5Phos/NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGCANN, where N are random bases.
6merFreq_neurons.tab
GFP.neu.A

data_processing:
Base calls were processed with bcl2fastq2 (Illumina) version 2.18.
51-nt or 68-nt raw reads that consisted of 3 random positions, a 4-nt multiplexing barcode, and another 2 random positions, followed by the actual read were initially trimmed to 45 nt from the 3'-end, and duplicates were discarded.
Reads were de-multiplexed (note that they are already de-multiplexed in this GEO record), and the random positions, barcode, and 3'-bases matching Illumina adaptors were removed.
Reads shorter than 25 nt were filtered out and remaining reads trimmed to 35 nt. These steps were carried out using Trimmomatic (Bolger et al., 2014). Surviving reads were mapped to the mouse genome/transcriptome (Ensembl annotation of NCBIm37) using tophat (Trapnell et al., 2009) with default settings. To prevent false assignments of reads from repetitive regions, any reads with a mapping quality < 3 were removed from further analysis.
The frequencies of all 6-mers within windows 50 nt of either side of the inferred likely crosslink site (the first base of the aligned read) on the genome sequence were calculated.
Genome_build: mm9
Supplementary_files_format_and_content: Tab delimited files with frequencies of 6-mers in windows 50 nt on either side of the inferred crosslink site of all reads

source_name: Cortical glutamatergic neurons differentiated in vitro from ES cells

extract_protocol:
Cells were crosslinked (0.15 J/cm2) at 254 nm with a Stratalinker 1800. Three independent replicates were used for generating iCLIP samples. Lysates generated from the crosslinked cells were treated with Turbo DNAse (Ambion) and diluted RNAse I (Ambion) for 5 minutes at 37°C to digest the genomic DNA and trim the RNA to short fragments of an optimal size range. RNA-protein complexes were immunoprecipitated using 100 μL of protein G Dynabeads (Life Technologies) and 10 μg of anti-Flag (Sigma) antibody. Following stringent high salt washes, the immunoprecipitated RNA was 3´ end dephosphorylated and a pre-adenylated adaptor was ligated at the 3´ end as described for a previously described enhanced CLIP ligation method (Van Nostrand et al., Nat. Methods 2016) for adaptor ligation. After additional washes, the immunoprecipitated RNA was 5´ end-labeled using radioactive 32P isotopes. The immunoprecipitated complexes were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Protran). RNA was recovered by digesting proteins using proteinase K.
RNA was reverse transcribed into cDNA using reverse transcription primers that include barcode sequences to enable multiplexing,  a unique molecular identifier, and a BamHI restriction site. The cDNA was size selected (low: 70 to 85 nt, middle: 85 to 110 nt, and high: 110 to 180 nt), circularized to add the adaptor to the 5′ end, digested at the internal BamHI site, and then PCR amplified using AccuPrime SuperMix I (Life Technologies). The final PCR libraries were purified by agarose gel electrophoresis using gel extraction columns (QIAGEN), eluted DNA was mixed at a ratio of 1:5:5 from the low, middle, and high fractions and submitted for sequencing.

treatment_protocol:
CGR8 ES cells were engineered to express Flag-tagged EGFP under the control of doxycyclin using the PiggyBac system, and single-cell subclones were derived. Cell were differentiated into cortical glutamatergic neurons as described previously (Hubbart et al., F1000 Res 2013). Neural precursor cells were dissociated and plated onto 15 cm plates coated with poly-D-Lysine and laminin (~20 million cells per plate). After 8 days Flag-EGFP expression was induced with 2 μg/ml doxycycline for 48 hours.
Crosslinking (0.15 J/cm2) was done at 254 nm with a Stratalinker 1800.

value: Neurons derived from CGR8 ES cells

value: ES-derived Cortical glutamatergic neurons

tag: antibody

value: Flag monoclonal antibody M2 (Sigma-Aldrich)


title: iCLIP-seq of Flag-Srrm4 in differentiated neurons, replicate 2

description:
Extracted RNA was reverse transcribed using primer Rt9clip: /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTGGCNN, where N are random bases.
6merFreq_neurons.tab
Srrm4.B

treatment_protocol:
CGR8 ES cells were engineered to express Flag-tagged Srrm4 under the control of doxycyclin using the PiggyBac system, and single-cell subclones were derived. Cell were differentiated into cortical glutamatergic neurons as described previously (Hubbart et al., F1000 Res 2013). Neural precursor cells were dissociated and plated onto 15 cm plates coated with poly-D-Lysine and laminin (~20 million cells per plate). After 8 days Flag-Srrm4 expression was induced with 2 μg/ml doxycycline for 48 hours.
Crosslinking (0.15 J/cm2) was done at 254 nm with a Stratalinker 1800.


title: Sox4KD2p1

city: Utrecht

first: Paul

last: Coffer

address: Uppsalalaan 6

department: Regenerative Medicine Center

country: Netherlands

institute: UMC Utrecht

data_processing:
Sequencing reads were mapped against the reference genome (mm10 assembly) using TopHat v2.0.9 as previously described (Trapnell et al., 2012). Only uniquely mapped reads were selected for further analysis.
guide transcripts were assembled using CuffLinks v2.2.1. Reads were quartile normalized using the –library-norm-method quartile option.
Differential gene analysis was performed using CuffDiff. Cluster 3.0
Genome_build: mm10
Supplementary_files_format_and_content: [gene_exp.diff.txt]  Tab-delimited text file containing  average differential gene expression FPKM normalized values (value_1 = SCR, value_2 = Sox4KD)

source_name: mouse E14.5 embryonic neural stem cells

growth_protocol: NSCs were growth as monolayer in poli-L-ornithine and laminin coated plates in presence of EGF and bFGF

extract_protocol:
total RNA-isolation with RNAeasy kit (Qiagen)
Sample preparation was performed using Poly(A)Purist MAG Kit (Thermo Scientific) according to manufacturers’ instructions. Isolated mRNA was subsequently repurified using mRNA-ONLY Eukaryotic mRNA Isolation Kit (Epicentre (Illumina, Inc.), Madison, WI, USA). Sequencing libraries were prepared using SOLiD Total RNA-Seq Kit (Applied Biosystems Life Technologies) according to the standard protocol recommendations

treatment_protocol: Upon lentiviral transduction NSCs were grown and expanded under puromycin selection before RNA-isolation (0.5 µg/mL)

tag: strain

value: embryonic neural stem cells

tag: cell source

value: R&D Systems, Minneapolis, MN

tag: shRNA

value: targeting Sox4

tag: shRNA name and source

value: TRCN0000012080, Sigma Aldrich, St. Louis, MS


title: Sox4KD2p2

tag: cell type


title: ARPE19_VitC_2_RNA

tag: treatment


title: ARPE19_Control_1_IP


title: SCRp2

value: scrambled

value: SHC002, Sigma Aldrich, St. Louis, MS


title: SCRp1


title: ARPE19_Control_2_RNA

value: ARPE-19 Cells


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 5

description:
Extracted RNA was reverse transcribed using primer Rt10clip: /5Phos/NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTCNN, where N are random bases.
6merFreq_N2A.tab
GFP.E

treatment_protocol:
EGFP was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced for 48 hours prior to crosslinking.
Crosslinking (0.15 J/cm2) was done at 254 nm with a Stratalinker 1800.

tag: cell line


title: Sox4KD1p2

value: TRCN0000012078, Sigma Aldrich, St. Louis, MS


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 6

description:
Extracted RNA was reverse transcribed using primer Rt14clip: /5Phos/NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGCANN, where N are random bases.
6merFreq_N2A.tab
GFP.F


title: iCLIP-seq of Flag-Srrm4 in differentiated neurons, replicate 1

description:
Extracted RNA was reverse transcribed using primer Rt1clip: /5Phos/NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTTNN, where N are random bases.
6merFreq_neurons.tab
Srrm4.A

growth_protocol: Cell were differentiated into cortical glutamatergic neurons as described previously (Hubbart et al., F1000 Res 2013).


title: Sox4KD1p1


title: TRAP_V1

city: St Louis

first: Joseph

middle: D

last: Dougherty

state: Missouri

address: 4566 Scott Ave, box 8232

department: Genetics and Psychiatry

institute: Washington University

data_processing:
RNA-seq reads were aligned to the Ensembl top-level assembly with STAR version 2.0.4b (Dobin et al. 2013) (RRID:SCR_015899).
Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Sequencing performance was assessed with RSeQC version 2.3 (L. Wang, Wang, and Li 2012) (RRID:SCR_005275) for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction, known junction saturation, and read distribution over known gene models.
Mouse: Ensembl76
Supplementary_files_format_and_content: Gene Counts

source_name: This sample is TRAP performed on the Vgat mouse

extract_protocol:
Five replicates of RBP4-TRAP and VGAT-TRAP were harvested by rapid forebrain dissection at 21 days post birth as described (Ouwenga et al. 2017; Westmark et al. 2011). Each replicate contained a pool of two to three forebrains of both sexes as available. Four samples were collected from each replicate in parallel: whole cell homogenate (WCH) was RNA isolated from an aliquot of the initial homogenization of the tissue, TRAP was the capture of GFP-tagged ribosomes from an aliquot of WCH. RNA isolated from a fraction of the WCH subjected to synaptoneurosomal fractionation (SNF), and SynapTRAP (ST) is TRAP performed on the SNF, as described (Ouwenga et al. 2017). RNA concentration for all was measured using a Nanodrop and diluted to <5ng/µL before being assessed for quality and concentration using an Agilent TapeStation 4200
Library preparation was performed with 30ng of total RNA from each sample. ds-cDNA was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech #634936) per manufacturer’s protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles/burst 50, time 120 seconds at 18 degrees. cDNA was blunt ended, had an A base added to the 3’ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 13 cycles using primers incorporating unique index tags.

value: Vgat TRAP

tag: tissue

value: Cortex


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 3

description:
Extracted RNA was reverse transcribed using primer Rt1clip: /5Phos/NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTTNN, where N are random bases.
6merFreq_N2A.tab
GFP.C


title: TRAP_V2


title: TRAP_V4


title: TRAP_R4

source_name: This sample is TRAP performed on the Rbp4 mouse

value: Rbp4 TRAP


title: TRAP_R5


title: TRAP_V3


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 2

description:
Extracted RNA was reverse transcribed using primer Rt16clip: /5Phos/NNTTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTTAANN, where N are random bases.
6merFreq_N2A.tab
GFP.B


title: TRAP_R3


title: RNA-Seq of N2A cells after knockdown of Rnps1, replicate 2


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 1

description:
Extracted RNA was reverse transcribed using primer Rt6clip: /5Phos/NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCCGGNN, where N are random bases.
6merFreq_N2A.tab
GFP.A


title: SNTRAP_V5

source_name: This sample is TRAP performed on the synaptoneurosome band from the Vgat mouse


title: iCLIP-seq of Flag-Srsf11 in N2A cells, replicate 2

description:
Extracted RNA was reverse transcribed using primer Rt13clip: /5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCGGANN, where N are random bases.
6merFreq_N2A.tab
Srsf11.B

treatment_protocol:
Srsf11 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced for 48 hours prior to crosslinking.
Crosslinking (0.15 J/cm2) was done at 254 nm with a Stratalinker 1800.


title: RNA-Seq of N2A cells after non-targeting knockdown (control for Rnps1 knockdown), replicate 1


title: TRAP_V5


title: iCLIP-seq of Flag-Srsf11 in N2A cells, replicate 3

description:
Extracted RNA was reverse transcribed using primer Rt16clip: /5Phos/NNTTAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTTAANN, where N are random bases.
6merFreq_N2A.tab
Srsf11.C


title: SNTRAP_V3


title: TRAP_R2


title: iCLIP-seq of Flag-GFP in N2A cells, replicate 4

description:
Extracted RNA was reverse transcribed using primer Rt1clip: /5Phos/NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNGGTTNN, where N are random bases.
6merFreq_N2A.tab
GFP.D


title: iCLIP-seq of Flag-Srsf11 in N2A cells, replicate 1

description:
Extracted RNA was reverse transcribed using primer Rt9clip: /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTGGCNN, where N are random bases.
6merFreq_N2A.tab
Srsf11.A


title: iCLIP-seq of Flag-Rnps1 in N2A cells, replicate 3

description:
Extracted RNA was reverse transcribed using primer Rt2clip: /5Phos/NNACAANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTTGTNN, where N are random bases.
6merFreq_N2A.tab
Rnps1.C

treatment_protocol:
Rnps1 was expressed in an N2A cell line engineered with the Flp-in system under the control of doxycyclin, and expression was induced for 48 hours prior to crosslinking.
Crosslinking (0.15 J/cm2) was done at 254 nm with a Stratalinker 1800.


title: SNTRAP_V2


title: SNTRAP_R5

source_name: This sample is TRAP performed on the synaptoneurosome band from the Rbp4 mouse


title: SNTRAP_V4


title: iCLIP-seq of Flag-Rnps1 in N2A cells, replicate 2

description:
Extracted RNA was reverse transcribed using primer Rt13clip: /5Phos/NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNCGGANN, where N are random bases.
6merFreq_N2A.tab
Rnps1.B


title: SNTRAP_R1


title: PreIPTrap_V5

source_name: This sample is a control that is cortex RNA, from a Vgat TRAP mouse


title: PreIPTrap_V1


title: PreIPTrap_V4


title: iCLIP-seq of Flag-Rnps1 in N2A cells, replicate 1

description:
Extracted RNA was reverse transcribed using primer Rt9clip: /5Phos/NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC. Reads start with 5'-NNNTGGCNN, where N are random bases.
6merFreq_N2A.tab
Rnps1.A


title: PreIPTrap_V3


title: PreIPTrap_R5

source_name: This sample is a control that is cortex RNA, from an RBP4 TRAP mouse


title: PreIPTrap_V2


title: TRAP_R1


title: PreIPTrap_R4


title: PreIPTrap_R3


title: SNTRAP_V1


title: PreIPTrap_R1


title: PreIPTrap_R2


title: PreIPSN_V5

source_name: This sample is the synaptoneurosome band of a sucrose percoll gradient without any TRAP, from a Vgat TRAP mouse.


title: SNTRAP_R4


title: SNTRAP_R3


title: PreIPSN_V4


title: PreIPSN_V3


title: zmbzip22_rep3

city: Beijing

first: Chaobin

last: Li

state: Beijing

address: 2 Yuanmingyuan West Road, NMICC Building

country: China

institute: China Agricultural University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed using fastx（version:0.0.13）, then mapped to B73 RefGen_v3.30 using TopHat（version:2.0.9）.
The unique reads were normalized as Reads Per Kilobase of exon model per Million mapped reads (RPKM) using samtools v0.1.19
Genome_build: Ensembl plants release-30 zea_mays
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

organism: Zea mays

source_name: kernel

extract_protocol:
RNA was extracted from pooled kernels at 15 DAP with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit.
RNA libraries were prepared for sequencing using standard Illumina protocols. Barcode for WT_rep1: GTCCGC, for WT_rep2: GTGAAA, for WT_rep3: ATCACGAT, for zmbzip22_rep1: CGTACG, for zmbzip22_rep2: GAGTGG, for zmbzip22_rep3: ACAGTGAT.

value: kernel

value: 15 DAP

tag: genotype

value: zmbzip22 homozygous mutant


title: zmbzip22_rep1


title: PreIPSN_V2


title: WT_rep3

value: Wild-type


title: PreIPSN_V1


title: WT_rep2


title: WT_rep1


title: PreIPSN_R5

source_name: This sample is the synaptoneurosome band of a sucrose percoll gradient without any TRAP, from an Rbp4 TRAP mouse.


title: PreIPSN_R4


title: zmbzip22_rep2


title: PreIPSN_R3


title: SNTRAP_R2


title: PreIPSN_R2


title: PreIPSN_R1


title: rep1-IgG

description:
rep1.txt
rep1.bed

data_processing:
CASAVA1.8.2 (Illumina) were used for image analysis and base calling.
Raw fastq data were trimmed using SolexaQA v2.5 program with Q20 (Phred score) and L40 (Length).
Cleaned data were then mapped to the maize reference genome using Bowtie2 with default parameters and allowing unique reads only.
Peak detection was done using MACS v2.0.10 using the indicated criteria.
UCSC bedGraph files were created at 1bp resolution and normalized to total alignable reads (reads-per-million).
Genome_build: Ensembl plants release-30 zea_mays
Supplementary_files_format_and_content: tab-delimited text files include contains information about called peaks.
Supplementary_files_format_and_content: bed files include the peak summits locations for every peaks.

source_name: Kernel

extract_protocol:
Endosperm Lysates were clarified from sonicated nuclei. ZmbZIP22-DNA complexes were isolated with antibody and IgG serves as antibody control
Libraries were prepared according to Illumina's instructions accompanying the TruSeq ChIP Sample Prep Kit (Catalog# IP-202-1012). ChIP DNA samples were subjected to end repair and A-base addition, followed by ligation with adapters. Barcode sequences of adapters correspond to the following: CTCAGA, ChIP of ZmbZIP22; GCGCTA, ChIP of IgG (for two replicates). After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~500 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. Illumina Genome Analyzer (GA) sequencing was performed by the OSUCCC Nucleic Acid Shared Resource-Illumina GA II Core.

value: Endosperm

value: wildtype

tag: chip antibody

value: IgG((Sigma, catalog# I5006, lot# SLBJ3775V, pack size 50MG))


title: rep2-IgG

description:
rep2.txt
rep2.bed


title: rep2-ZmbZIP22

description:
rep2.txt
rep2.bed

value: anti-ZmbZIP22 (produced by submitter)


title: rep1-ZmbZIP22

description:
rep1.txt
rep1.bed


title: colorectal_AOM/DSS treatment_2w_rep2

city: Chengdu

first: Hongxin

last: Deng

state: Sichuan

address: Ke-yuan Road 4, No. 1, Gao-peng Street

department: West China Hospital

institute: Sichuan University and Collaborative Innovation Center for Biotherapy

description:
inflammation
gene expression after 2 week AOM/DSS treatment in colorectal tissues

hyb_protocol: 0.6 μg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 22.5μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 22.5μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 13.0(Agilent). Probe that at least 1 out of 2 samples flagged as Detected were maintained.   Probe sequences for non-control probes are included in the raw data files.

source_name: colorectal tissue, AOM/DSS, 2 week, replicate 2

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: C57 mice (20 g weight, male) were purchased from Beijing HFK Bioscience Co. (Beijing, China) and housed in a specific-pathogen free (SPF) room (25 ˚C) under a 12-h light/dark cycle with ad libitum access to food and water.

extract_protocol: RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556 ) following the manufacturer's instructions.

treatment_protocol: Mice were injected intraperitoneally (i.p.) with 10 mg/kg azoxymethane (AOM; Sigma-Aldrich, Germany). After 7 days, the mice received drinking water containing 2% DSS for 7 days followed by normal drinking water for 14 days. Totally, three DSS cycles were performed.

tag: background strain

tag: Sex

value: male

value: colorectal

tag: diagnosis

value: colitis


title: colorectal_AOM/DSS treatment_2w_rep1

source_name: colorectal tissue, AOM/DSS, 2 week, replicate 1


title: colorectal_AOM/DSS treatment_10w_rep1

description:
tumor
gene expression after 10 week AOM/DSS treatment in colorectal tissues

source_name: colorectal tissue, AOM/DSS, 10 week, replicate 1

value: colorectal tumor


title: DMS-treated SOD1 yeast library grown in YPD Biological Replicate B

status: Public on Oct 11 2018

city: Boston

first: Zarmik

last: Moqtaderi

state: MA

address: 240 Longwood Avenue C2-355

department: BCMP

institute: Harvard Medical School

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_SOD1_Library_RepB_NetReadsByIsoform.txt [Tab-delimited text file containing read counts in treated and control samples (as well as net read counts) for every 3' isoform]

organism: Saccharomyces cerevisiae

source_name: Yeast cells

growth_protocol: Cells were cultured in YPD medium to mid-log phase prior to treatment and harvesting

extract_protocol:
Hot acidic phenol extraction of total RNA, followed by purification on Qiagen Rneasy columns with Dnase I treatment
Hybridization capture and library construction: 500 ng of pooled barcoded DREADS libraries were blocked with 5 mg Cot-1 DNA (Invitrogen) and 1 ml each of p5 and p7 custom blocker small RNA (IDT).  Hybridization of a locked nucleic acid probe (5’-/5Biosg/AGTAAGCGCAT+CAATT+CAT+TC-3’) specific to SOD1 (Exiqon) was carried out using the xGen hybridization and wash kit (IDT) according to the manufacturer’s instructions, except that the hybridization and was carried out at 55˚C for 18 hours.  Subsequent capture, washes and purification were performed exactly as described in the kit’s user manual, with the exception of all the steps that require incubation at 65˚C were done at 55˚C.  Captured fragments were eluted from the beads in 50 ml EB for 5 minutes 75˚C and amplified by 15 cycles of PCR with Illumina p5 and p7 primers.  Libraries were purified by 8% PAGE and subjected to deep sequencing using the DREADS/SHREADS library protocol as described above.

treatment_protocol:
We constructed a plasmid library containing the SOD1 genomic locus (from 500 nt upstream of the ATG to 400 nt downstream of the stop codon) randomized in the 3’ UTRs at positions +66 to +70 relative to the stop codon.  The library was transformed into S. cerevisiae JGY2000, and the transformants were grown selectively in casamino acid medium lacking uracil, diluted into 50 ml of YPD and grown at 30˚C to OD600 = 0.5.  DMS-treated sample (added DMS to 1/30 v/v for 2 min at RT; quenched twice  with 2 volumes stop solution consisting of 30% 2-mercaptoethanol and 25% isoamyl alcohol)
Some samples were treated with dimethyl sulfate or NAI as described in the methods

tag: library strategy

value: DREADS


title: colorectal_AOM/DSS treatment_10w_rep3

source_name: colorectal tissue, AOM/DSS, 10 week, replicate 3


title: Untreated control SOD1 yeast library grown in YPD Biological Replicate B

description: JGY2000_SOD1_Library_RepB_NetReadsByIsoform.txt

treatment_protocol:
no-DMS control
We constructed a plasmid library containing the SOD1 genomic locus (from 500 nt upstream of the ATG to 400 nt downstream of the stop codon) randomized in the 3’ UTRs at positions +66 to +70 relative to the stop codon.  The library was transformed into S. cerevisiae JGY2000, and the transformants were grown selectively in casamino acid medium lacking uracil, diluted into 50 ml of YPD and grown at 30˚C to OD600 = 0.5.
Some samples were treated with dimethyl sulfate or NAI as described in the methods


title: JGY2000 grown in YPD NAI-treated SHREADS Biological Replicate A

description: 2 sets of fastq files because more was sequenced in a second run for extra depth

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_A_NAI_BothRunsCombined_SHREADS_5'endfreqs_Normed_1.2xTo65M.txt [Tab-delimited text file containing tabulated frequencies of all RT endpoints for every 3' isoform (consolidated both sequencing runs for this rep)]

extract_protocol:
Hot acidic phenol extraction of total RNA, followed by purification on Qiagen Rneasy columns with Dnase I treatment
DREADS/SHREADS library protocol: 25 µg mRNA was purified from total RNA samples with oligo dT(25) magnetic beads (NEB), and was then bound to beads pre-coated with a U45T5 biotinylated mRNA capture oligonucleotide1.  Immobilized mRNA tails were trimmed with RNase H to generate shortened poly(A) sequences averaging ~ 5 A’s as described1.  After ligation of 5’ pre-adenylated adapter A to the trimmed mRNA 3’ ends, mRNAs were reverse transcribed with Superscript III (Life Technologies) using primer B1.  cDNAs were ligated with T4 DNA Ligase to splint adapter C, and the single-stranded DNA products (110 – 500 nt) were purified on 6% polyacrylamide-urea gels.  DNA fragments were amplified by 18 cycles of PCR with barcoded oligonucleotides as described1.  Amplified libraries were purified with SizeSelector-I beads (Aline Biosciences) as described1. Adapter A: 5′-/5rApp/NNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC/-3′  Primer B: 5′ -GTTCAGAGTTCTACAGTCCGACGATC-3′  Splint Adapter: This is a double-stranded adapter where Z is the double-stranded equivalent of N.                                      5’ – ZZZZTGGAATTCTCGGGTGCCAAGG – 3’                     3’ – NNNNNNZZZZACCTTAAGAGCCCACGGTTCC – 5’  1. Jin, Y. et al. Mapping 3' mRNA isoforms on a genomic scale. Curr. Protoc. Mol. Biol. 110, 4.23.21-24.23.17 (2015)

treatment_protocol:
NAI treatment: 25 ml cells were resuspended in a total volume of 380ml of pre-warmed YPD.  Cells were incubated with either 20ml of 2M NAI (in DMSO; +NAI sample) or with 20ml DMSO (-NAI control) at 30˚C for ~ 3 minutes, centrifuged and washed twice with water prior to freezing at -80˚C.
Some samples were treated with dimethyl sulfate or NAI as described in the methods

value: SHREADS


title: colorectal_AOM/DSS treatment_10w_rep2

source_name: colorectal tissue, AOM/DSS, 10 week, replicate 2


title: colorectal_without treatment_0w_rep3

description:
normal
gene expression of colorectal tissue

source_name: colorectal tissue, AOM/DSS, 0 week, replicate 3

value: normal colorectal tissue


title: JGY2000 grown in YPD NAI-treated SHREADS Biological Replicate B

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_B_NAI_BothRunsCombined_SHREADS_5'endfreqs_Normed_0.838xTo65M.txt [Tab-delimited text file containing tabulated frequencies of all RT endpoints for every 3' isoform (consolidated both sequencing runs for this rep)]


title: colorectal_AOM treatment_1w_rep3

description:
inflammation
gene expression after 1 week AOM treatment in colorectal tissues

source_name: colorectal tissue, AOM, 1 week, replicate 3


title: JGY2000 grown in YPD untreated control SHREADS Biological Replicate B

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_B_DMSO_BothRunsCombined_SHREADS_5'endfreqs_Normed_0.906xTo65M.txt [Tab-delimited text file containing tabulated frequencies of all RT endpoints for every 3' isoform (consolidated both sequencing runs for this rep)]

treatment_protocol:
no-NAI control (DMSO only)
Some samples were treated with dimethyl sulfate or NAI as described in the methods


title: colorectal_AOM/DSS treatment_2w_rep3

source_name: colorectal tissue, AOM/DSS, 2 week, replicate 3


title: colorectal_AOM treatment_1w_rep2

source_name: colorectal tissue, AOM, 1 week, replicate 2


title: DMS-treated SOD1 yeast library grown in YPD Biological Replicate A

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_SOD1_Library_RepA_NetReadsByIsoform.txt [Tab-delimited text file containing read counts in treated and control samples (as well as net read counts) for every 3' isoform]


title: Untreated control SOD1 yeast library grown in YPD Biological Replicate A

description: JGY2000_SOD1_Library_RepA_NetReadsByIsoform.txt


title: B1

city: harbin

first: Lishuang

last: Qi

address: No. 157, Baojian Road, Nangang District

department: College of Bioinformatics Science and Technology

institute: Harbin Medical University

description: lung squamous cell carcinoma

hyb_protocol: Total RNAs were hybridized using mRNA + lncRNA Human Gene Expression Microarray V4.0 (4×180K format,  CapitalBio Corp, Beijing, China)

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities.  Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: lung squamous cell carcinoma

label_protocol: The purity and concentration of RNA was determined by Nano Drop ND-1000 spectrophotometer according to OD260/280 reading.

extract_protocol: Tumor samples were microdissected to ensure the purity of the tumor.

treatment_protocol: Samples were fresh frozen and were shipped on ice for subsequent RNA extraction

tag: gender

value: M

tag: age

tag: tnm

value: T1aN0M0 Ⅰa


title: colorectal_AOM treatment_1w_rep1

source_name: colorectal tissue, AOM, 1 week, replicate 1


title: B3

description: lung squamous cell carcinoma


title: JGY2000 grown in YPD untreated control SHREADS Biological Replicate A

data_processing:
python scripts: remove 4 random nt from each read, count and remove initial Ts from R1 reads, discard any reads containing Ns, truncate remainder of each read to 17nt max (starting at nt 2 for the R2 side), discard reads with fewer than 9nt remaining after T removal.
bowtie: map to genome using bowtie with one mismatch and m1, X1000
python: screen the mapped reads to determine which initial Ts arose from genomically encoded As vs As added after cleavage
Microsoft Excel: normalize to equal number of total processed reads per sample
python: readjust mapped first position of R2 back 2nt: one nt to compensate for having started mapping from nt 2, and one more because the potential DMS-modified nt is 1 nt upstream of the last reverse-transcribed position
python: For DREADS samples, consolidate instances of every possible DMS-modfied position (mapped R2 read start position minus 2) for every 3' isoform
python: For every isoform, compare A and C positions of DMS-treated and control samples, subtracting read count of control from DMS sample to obtain net reads for the position
python: Obtain Winsorized reactivity values for every isoform by averaging the top 15 net reads values in the 300-nt window, assigning this averaged top value a score of 1, and scaling all other net reads values relative to this.
RNAStructure: predict folding of mRNA isoforms (assuming a 30-nt poly(A) tail) with and without considering the DMS reactivity values. (parameters: 30°C, 200 nt maximum pairing window)
Genome_build: S. cerevisiae build SacCer 3;  S. pombe build EF2; D. hansenii Deha2; K. lactis Klla0
Supplementary_files_format_and_content: Tab-delimited text files containing consolidated reads for each 3' isoform
Supplementary_files_format_and_content: JGY2000_A_DMSO_BothRunsCombined_SHREADS_5'endfreqs_Normed_1.143xTo65M.txt [Tab-delimited text file containing tabulated frequencies of all RT endpoints for every 3' isoform (consolidated both sequencing runs for this rep)]


title: ZEB1KO25D

first: Xiangxiang

last: Jiang

address: 1 Beichen West Road, Chaoyang District

institute: Institute of Zoology, Chinese Academy of Sciences

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence to get the clean reads
Then the clean reads were mapped to hg38 whole genome using HISAT2
FPKM and Count values were calculated using StringTie and Ballgown
Genome_build: hg38
Supplementary_files_format_and_content: xlsx file includes RPKM values and count values for each Sample.

source_name: Differentiated hESCs

growth_protocol: hESCs were cultured during routinely protocol

extract_protocol:
Medium was removed, cells were washed with PBS, and RNA was harvested using Trizol reagent.
The sequencing library for Illumina HiSeq 4000 sequencer was constructed with 0.2 μg of total RNA ofeach sample by PE150 strategy.

value: inner cell mass

value: ESCs differentiate neural cells

tag: passage

tag: differentiation day


title: B2

description: lung squamous cell carcinoma

value: T2aN0M0 Ⅰb

tag: smoke


title: ZEB1KO30D


title: colorectal_without treatment_0w_rep1

source_name: colorectal tissue, AOM/DSS, 0 week, replicate 1


title: colorectal_without treatment_0w_rep2

source_name: colorectal tissue, AOM/DSS, 0 week, replicate 2


title: ZEB1KO10D


title: ZEB1KO15D


title: UB_Input_DNA

city: North Hills

first: Masaki

last: Nishikawa

address: 16111 Plummer st

institute: UCLA / VA

data_processing:
For ChIP-seq analysis, the replicate was concatenated and mapped using Bowtie2 at default setting.
ChIP-seq peaks were called using MACS14 against the input control data. The parameter set used in MACS14 was as follows: H3K4me3 (Band width 300, Pvalue cutoff 1e-05, the nearby regions to calculate dynamic lambda 1000-10000) and H3K27me3 (Band width 2900, Pvalue cutoff 1e-04 (mES and MM(Cited1)) or 1e-03 (UB), the nearby regions to calculate dynamic lambda 1000-20000).
Peaks were annotated with overlapping genes using AnnotateGenomicRegions (  https://sourceforge.net/projects/annotatelocus/files/webarchive/JSFversion/AnnotateGenomicRegions.war/download)
For RNA-seq analysis, all sequencing reads were mapped using Tophat at default setting.
Aligned RNA-seq reads were further analyzed using Cufflinks, Cuffmerge, and Cuffdiff
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-seq: BED files with called peaks on sample records and peak annotation files on series record.
RNA-seq: text file with FPKM

source_name: Ureteric epithelium

extract_protocol: Unfixed cells were lysed and treated with Micrococcal nuclease. Libraries were prepared using ThruPLEX-FD or ThruPLEX DNA-seq kit (Rubicon Genomics)

value: Ureteric epithelium

value: Hoxb7-GFP+ ureteric epithelium

tag: strain

value: (C57BL/6J x CBA/J)F2 origin, maintained with C57BL/6J background

value: Hoxb7myr- Venus/+


title: UB_H3K27me3_ChIPSeq

tag: cell type

value: anti-H3K27me3 (Active motif, #39157)


title: WT35d

value: WT


title: ZEB1KO35D


title: MM_Input_DNA

source_name: Metanephric mesenchyme (cap mesenchyme)

value: Metanephric mesenchyme (cap mesenchyme)

value: Cited1-RFP+ cap mesenchyme

value: (C57BL/6 x DBA)F1 origin, maintained with C57BL/6J background (N5)


title: UB_H3K4me3_ChIPSeq

value: anti-H3K4me3 (Abcam, ab8580)


title: WT30d


title: MM_H3K4me3_ChIPSeq


title: MM_H3K27me3_ChIPSeq


title: WT25d


title: WT15d


title: WT10d


title: PA14 rep3

city: kunming

first: Yi-Cheng

last: Ma

email: 290361200@qq.com, mayc@ynu.edu.cn

state: yunnan

address: NO.2 North Cuihu Road

institute: Yunnan University

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing poly-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using TopHat v2.0.12. We selected TopHat as the mapping tool for that TopHat can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels (Trapnell, Cole, et al., 2010).
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate . Genes with an adjusted P-value.The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.005 and log2(Fold change) of 1 were set as the threshold for significantly differential expression.
Genome_build: Caenorhabditis elegans WBcel235
Supplementary_files_format_and_content: txt files include FPKM values for each Sample

organism: Caenorhabditis elegans

source_name: worms with P. aeruginosa infection

growth_protocol: Worms were maintained on nematode growth media (NGM) and fed with E. coli OP50 at 20°C

extract_protocol:
Bacteria were removed and worms were natural precipitation by M9 buffer , flash frozen on dry ice, and RNA was harvested using Trizol reagent.A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: P. aeruginosa or E. coli OP50  was incubated first for 24 h at 37ºC and then for 24 h at 25ºC.  Young adult animals were transferred onto the plates at 25°C and harvesting worms after 24 hours.

tag: developmental stage

value: Adult worms

value: wild type


title: PA14 rep2


title: PA14 rep1


title: OP50 rep2

source_name: worms fed E. coli OP50


title: OP50 rep1


title: mES_H3K27me3_ChIPSeq

source_name: mouse embryonic stem cells

value: mouse embryonic stem cells

value: Passage 15


title: mES_H3K4me3_ChIPSeq


title: WT

first: Shengde

last: Liu

address: 38 Xueyuan Rd , Haidian District

institute: Peking University Health Science Center

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
FPKM were calculated. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: mm8
Supplementary_files_format_and_content: FPKM values for each Sample

source_name: macrophage

extract_protocol:
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: immortalized bone marrow-derived macrophages (iBMDMs)


title: WT-HSV-1

description: WT HSV-1_combine

tag: infected with

value: HSV-1 for 6 h


title: OP50 rep3


title: mES_Input_DNA

tag: tissue


title: AGO2 IP, triptolide treatment, replicate 2

city: Bethesda

first: Cameron

last: Palmer

address: 50 South Drive

department: National Institute of Diabetes and Digestive and Kidney Diseases

institute: National Institutes of Health

data_processing:
Illumina HiSeq 2500 sequencing, 50bp reads.
Adapter removal with cutadapt v1.10
Alignment to Drosophila melanogaster genome with bowtie2
Removal of duplicates with picard v2.11.0 MarkDuplicates
Narrow peak calling with MACS2 v2.1.1.20160309 callpeak
Genome_build: dm6
Supplementary_files_format_and_content: Coverage tracks in bigWig format, normalized to RPGC. For IP samples, bed format peak call output, from the narrowPeak output of macs2.

organism: Drosophila melanogaster

source_name: embryo

growth_protocol: Kc167 cells were grown in CCM3 media (Thermo Scientific HyClone). Cells were maintained at 25°C.

extract_protocol:
Approximately 1-3 X 10^7 cells were fixed by addition of 1% formaldehyde to cell media for 10 min at RT with gentle agitation. Formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. Cells were pelleted at 2000 xg, washed twice in PBS, and resuspended in 0.8 mL ice–cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) supplemented with Complete protease inhibitors (Roche), incubated on ice 10 min, and pelleted by centrifugation at 2000 xg for 5 min at 4°C. Next, the supernatant was removed and pellets were resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA.Na2, 1% SDS) and incubated for 10 min at 4°C. Afterwards, 0.5 mL of IP dilution buffer was added (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS), and chromatin was fragmented to an average size of 300 bp by using PicoBioruptor (Diagenode) using 10 cycles of 30 s on plus 30 s off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at -80°C. Chromatin was diluted to 1:5 with IP buffer, and the assayed antibody in addition to 50 μL of prewashed protein A/G 50% slurry was added and rotated overnight at 4°C. The next day, beads were washed first three times with low salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS); next three times with high salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS); finally two times with LiCl buffer (10 mM Tris-HCl pH 8, 1 mM EDTA.Na2, 250 mM LiCl, 1% NP-40, 1% DOC). Chromatin was eluted twice with 200 μL of elution buffer (500 μL of 1M NaHCO3, 250 μL of 20% SDS, 4.25 mL dH2O) for 30 min at 65°C each and further incubated overnight at 65°C with 38 μL of decrosslinking solution (20 μL of 5M NaCl, 8 μL of 0.5M EDTA, 10 μL of 1M Tris-HCl pH 8). After de-crosslinking, samples were treated with Proteinase K for 2 h at 50°C and then combined with 1 vol of phenol/chloroform/isoamyl alcohol (25:24:1), vortexed 15 s, and centrifuged for 5 min at 10,000 xg . The top layer was transferred to a new tube, and the procedure was repeated using 1 vol chloroform. The top layer was collected and subsequently precipitated with 0.1 vol of 3M NaOAc pH 5.2 and 2.5 vol of 100% ethanol supplemented with 2 μL of Glycoblue (Ambion). After incubating 30 min at -80°C, samples were centrifuged 20 min at 4°C at 10,000 xg. Pellets were washed with 70% ethanol and centrifuged 5 min at 4°C at 10,000 xg. Pellets were air dried at RT prior to resuspension in 10 μL of dH2O.
Samples for ChIP-seq were prepared according to the manufacturer’s protocol with TruSeq adapters (Illumina).

treatment_protocol: Cells were treated with Triptolide 10 μM for 60 min.

tag: cell line

value: female

tag: treatment

value: triptolide

value: AGO2 (Mueller Lab)


title: AGO2 IP, mock treatment, replicate 1

value: mock

tag: chip antibody


title: ChRIP-seq, input of AGO2 IP, replicate 1

data_processing:
Illumina HiSeq 2500 sequencing, 50bp reads.
Adapter removal with cutadapt v1.15
Alignment to Drosophila melanogaster genome with hisat v2.1.0
Assignment of reads to gene features with Subread featureCounts v1.5.3
Signal detection via differential expression analysis between inputs and IP samples with DESeq2 1.18.1
Genome_build: dm6
Supplementary_files_format_and_content: Number of reads assigned to gene features, as computed by Subread featureCounts v1.5.3

extract_protocol:
Approximately 1-3 X 10^7 cells were fixed by addition of 1% formaldehyde to cell media for 10 min at RT with gentle agitation. Formaldehyde was quenched by addition of glycine to 0.125 M with gentle agitation for 5 min at RT. Cells were pelleted at 2000 xg, washed twice in PBS, and resuspended in 0.8 mL ice–cold cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) supplemented with Complete protease inhibitors (Roche) and RNasin (Promega), incubated on ice 10 min, and pelleted by centrifugation at 2000 xg for 5 min at 4°C. Next, the supernatant was removed and pellets were resuspended in 1 mL nuclear lysis buffer (50 mM Tris-HCl pH 8, 10 mM EDTA.Na2, 1% SDS, RNasin (Promega)) and incubated for 10 min at 4°C. Afterwards, 0.5 mL of IP dilution buffer was added (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS), and chromatin was fragmented to an average size of 300 bp by using PicoBioruptor (Diagenode) using 10 cycles of 30 s on plus 30 s off, maximum output. Samples were centrifuged at max speed for 10 min at 4°C, and the supernatant (sheared chromatin) was saved at -80°C. Chromatin was diluted to 1:5 with IP buffer, and the assayed antibody in addition to 50 μL of prewashed protein A/G 50% slurry, preblocked for one hour with 0.5% of tRNA and 0.2% BSA, was added and rotated overnight at 4°C. The next day, beads were washed first three times with low salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS); next three times with high salt IP dilution buffer (20 mM Tris-HCl pH 8, 2 mM EDTA.Na2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS); finally two times with LiCl buffer (10 mM Tris-HCl pH 8, 1 mM EDTA.Na2, 250 mM LiCl, 1% NP-40, 1% DOC). After the last LiCl wash, beads were resuspended in 100 μL of 50mM Tris-Cl pH 7.0, 5 mM EDTA and 1% SDS and de-crosslinked at 65°C for 2 h. Next, beads were treated with DNAse I for 15 min at 37°C, and total RNA was extracted with TRIzol (Invitrogen).
RNA-seq libraries were prepared with Ovation RNA-seq Systems 1-16 for Model Organisms (Nugen).

tag: Sex


title: input of AGO2 IP, triptolide treatment, replicate 1


title: ChRIP-seq, AGO2 IP, replicate 1

value: AGO2 (Mueller lab)


title: AGO2 IP, mock treatment, replicate 2


title: ChRIP-seq, input of AGO2 IP, replicate 2


title: ChRIP-seq, AGO2 IP, replicate 3


title: input of AGO2 IP, triptolide treatment, replicate 2


title: Anti-miR-223, biological rep2

city: AMIENS

first: Laurent

last: METZINGER

address: Avenue René Laënnec

country: France

institute: INSERM U1088

description: The cells will pinocytose neutral red and will phagocytose latex beads and zymosan. They are capable of antibody dependent lysis of sheep erythrocytes and tumor cell targets. LPS or PPD treatment for 2 days stimulates lysis of erythrocytes but not tumor cell targets.

hyb_protocol: Hybridization was performed on Gene Atlas array strips (Mouse Gene 2.1 ST Array strip) at 48°C for 18 hours on the Affymetrix GeneAtlas Hybridization Station.

scan_protocol: Strips were scanned using the Gene Atlas imaging station.

data_processing: Data were normalized using the Expression Console software (Affymetrix) using default RMA-sketch normalization. Normalized .chp files were then analyzed using Partek Genomic Suite (Partek) using default settings.

label: biotin

source_name: RAW 264.7 cells were  transfected for 48 h with anti-miR-223 (Qiagen)

label_protocol: Labeling was performed according to the GeneChip WTPLUS Reagent kit protocol

growth_protocol: Cell culture was performed in the same conditions for all techniques used. First, RAW 264.7 cells were seeded in 6-well culture plates(Corning) or Petri Dish (10mm2) at 10,000 cells/cm2, grown until confluency

extract_protocol: For the transcriptomic analysis, pellet was suspended and homogenized in the relevant extraction buffer (miRNeasy kit, Qiagen) and RNA was extracted according to manufacturer's instructions. The yield of the extracted RNA was determined spectrophotometrically (Nanoview) by measuring the optical density at 260 nm. RNA quality was evaluated using the RNA LabChip and Agilent 2100 Bioanalyzer

value: macrophage; Abelson murine leukemia virus transformed

value: RAW 264.7 adherent monocyte/macrophage cell line

value: transfected with anti-miR-223


title: Pre-miR-223, biological rep1

source_name: RAW 264.7 cells were  transfected for 48 h with pre-miR-223 (Qiagen)

value: transfected with pre-miR-223


title: Anti-miR-223, biological rep1


title: Anti-miR-223, biological rep4


title: Scramble, biological rep2

source_name: RAW 264.7 cells were  transfected for 48 h with irrelevant scramble (Sbl)

value: transfected with scramble


title: input of AGO2 IP, mock treatment, replicate 2


title: Anti-miR-223, biological rep3


title: Pre-miR-223, biological rep4


title: Pre-miR-223, biological rep2


title: Scramble, biological rep1


title: input of AGO2 IP, mock treatment, replicate 1


title: Pre-miR-223, biological rep3


title: Scramble, biological rep4


title: input of RNA PolII S5 IP, dsLaminB treatment, replicate 1

data_processing:
Illumina HiSeq 2500 sequencing, 50bp reads.
Adapter removal with cutadapt v1.10
Alignment to Drosophila melanogaster genome with bowtie2
Removal of duplicates with picard v2.11.0 MarkDuplicates
Broad peak calling with MACS2 v2.1.1.20160309 callpeak --broad
Genome_build: dm6
Supplementary_files_format_and_content: Coverage tracks in bigWig format, normalized to RPGC. For IP samples, bed format peak call output, from the broadPeak output of macs2.

treatment_protocol: Amplicons used for dsRNA knockdowns were designed based on recommendations from the Drosophila RNAi Screening Center. Templates were PCR-amplified from genomic DNA using primers containing the T7 promoter sequence. In vitro transcription of PCR templates using the MEGAscript T7 kit (Ambion) was used to produce dsRNAs, and these were purified by phenol-chloroform extraction. Transfections using 2 μg of dsRNA or 100 pmol of siRNA, or no dsRNA/siRNA as mock treatment, were performed using Cell Line Nucleofector kit V (Amaxa Biosystems) transfection reagent using the G-30 program. Three days after transfection, cells were collected and knockdown efficiency was confirmed by Western blotting.

value: dsLaminB

tag: genotype

value: LaminB knockdown


title: input of RNA PolII S5 IP, dsLaminB treatment, replicate 2


title: ChRIP-seq, input of AGO2 IP, replicate 3


title: input of RNA PolII S5 IP, siAGO2 treatment, replicate 2

value: Ago2 knockdown


title: input of RNA PolII S5 IP, siAGO2 treatment, replicate 1


title: AGO2 IP, triptolide treatment, replicate 1


title: input of RNA PolII S5 IP, mock treatment, replicate 2

value: control


title: Scramble, biological rep3


title: RNA PolII S5 IP, dsLaminB treatment, replicate 1

value: RNA PolII S5 (Abcam Ab5408)


title: input of RNA PolII S5 IP, mock treatment, replicate 1


title: RNA PolII S5 IP, dsLaminB treatment, replicate 2


title: input of RNA PolII S2 IP, dsLaminB treatment, replicate 2


title: RNA PolII S5 IP, siAGO2 treatment, replicate 2


title: RNA PolII S5 IP, mock treatment, replicate 2


title: input of RNA PolII S2 IP, mock treatment, replicate 2


title: input of RNA PolII S2 IP, siAGO2 treatment, replicate 1


title: RNA PolII S5 IP, siAGO2 treatment, replicate 1


title: input of RNA PolII 8WG16 IP, dsLaminB treatment, replicate 2

data_processing:
Illumina HiSeq 2500 sequencing, 50bp reads.
Adapter removal with cutadapt v1.10
Alignment to Drosophila melanogaster genome with bowtie2
Removal of duplicates with picard v2.11.0 MarkDuplicates
Broad peak calling with MACS2 v2.1.1.20160309 callpeak –broad
Genome_build: dm6
Supplementary_files_format_and_content: Coverage tracks in bigWig format, normalized to RPGC. For IP samples, bed format peak call output, from the broadPeak output of macs2.


title: input of RNA PolII 8WG16 IP, dsLaminB treatment, replicate 1


title: RNA PolII S2 IP, siAGO2 treatment, replicate 2

value: RNA PolII S2 (Abcam Ab5095)


title: RNA PolII S2 IP, mock treatment, replicate 1


title: input of RNA PolII S2 IP, mock treatment, replicate 1


title: RNA PolII S2 IP, mock treatment, replicate 2


title: input of RNA PolII 8WG16 IP, siAGO2 treatment, replicate 2


title: RNA PolII S2 IP, dsLaminB treatment, replicate 2


title: RNA PolII S5 IP, mock treatment, replicate 1


title: RNA PolII S2 IP, dsLaminB treatment, replicate 1


title: RNA PolII 8WG16 IP, siAGO2 treatment, replicate 2

value: RNA PolII 8WG16 (Biolegend 920102)


title: RNA PolII 8WG16 IP, dsLaminB treatment, replicate 2

value: female


title: input of RNA PolII 8WG16 IP, mock treatment, replicate 2


title: RNA PolII S2 IP, siAGO2 treatment, replicate 1


title: RNA PolII 8WG16 IP, dsLaminB treatment, replicate 1


title: input of RNA PolII S2 IP, dsLaminB treatment, replicate 1


title: RNA PolII 8WG16 IP, siAGO2 treatment, replicate 1


title: RNA PolII 8WG16 IP, mock treatment, replicate 2


title: input of RNA PolII 8WG16 IP, mock treatment, replicate 1


title: input of RNA PolII S2 IP, siAGO2 treatment, replicate 2


title: input of NELF-E IP, siAGO2 treatment, replicate 2


title: input of NELF-E IP, siAGO2 treatment, replicate 1


title: input of RNA PolII 8WG16 IP, siAGO2 treatment, replicate 1


title: NELF-E IP, siAGO2 treatment, replicate 1

value: NELF-E (Gilmour Lab)


title: NELF-E IP, siAGO2 treatment, replicate 2


title: input of CDK9 IP, siAGO2 treatment, replicate 2


title: NELF-E IP, mock treatment, replicate 2


title: NELF-E IP, mock treatment, replicate 1


title: input of CDK9 IP, siAGO2 treatment, replicate 1


title: input of CDK9 IP, mock treatment, replicate 2


title: input of CDK9 IP, mock treatment, replicate 1


title: CDK9 IP, siAGO2 treatment, replicate 2

value: CDK9 (Nakamura Lab)


title: CDK9 IP, siAGO2 treatment, replicate 1


title: RNA PolII 8WG16 IP, mock treatment, replicate 1

value: mock


title: CDK9 IP, mock treatment, replicate 2


title: CDK9 IP, mock treatment, replicate 1


title: input of NELF-E IP, mock treatment, replicate 1


title: input of NELF-E IP, mock treatment, replicate 2


title: F-RIG-I fRIP, replicate2

city: Nashville

first: Jeff

last: Rathmell

state: TN

address: 1211 Medical Center Drive

department: PMI

institute: Vanderbilt University Medical Center

description: fRIP RNA

data_processing:
The fastq raw reads were evaluated using fastqc
Then the reads were mapped to GRCh38 p10 reference genome using STAR_2.5.3a
The the aligned reads were assembled using gencode.v27 annotation with cufflinks _2.2.1
Then the enrichment of features were analysis with cuffquant and cuffdiff.
Genome_build: GRCh38.p10; GQ994935 (HHV8 genome)
Supplementary_files_format_and_content: cuffdiff output in plain text

source_name: patient-derived PEL cell line

extract_protocol:
BC-3 cells were crosslinked with 1% formaldehyde for 10 min in PBS, and unreacted formaldehyde was neutralized with 0.3 M glycine, pH 7, for 5 min. Cells were washed 2X with PBS, then resuspended in RIPA buffer (50 mM Tris, pH 8, 1% IGEPAL CA 630, 0.5% sodium deoxycholate, 0.05% SDS, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, and RNAse and protease inhibitors) and kept on ice for 10 min. Soluble cell extracts were incubated with anti-FLAG M2 magnetic resin at 4 °C for 2 h. Resin was washed three times for 10 min and then two times for 5 min at room temperature in RIPA buffer containing 0.1% SDS, 1 M NaCl, and 1 M urea. Resin was eluted with 1xFLAG peptide in RIPA buffer for 45 min at 4 °C. Protein–RNA crosslinks were reversed by adding 100 mM Tris, pH 8, 10 mM EDTA, 1% SDS, and 2 mM DTT to eluted samples and heating to 70 °C for 45 min. RNA was recovered by extraction with TRIzol and then again with phenol:chloroform:isoamyl alcohol [25:24:1 (vol/vol)] followed by ethanol precipitation.
Paired-end RNA-sequencing libraries were prepared from the recovered RNA using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) according to the manufacture recommendations. Libraries were then subjected to paired-end sequencing on a HiSeq3000 with 150 cycles at the Vanderbilt Technologies for Advanced Genomics (VANTAGE).

value: inducible F-RIG-I BC-3 cells


title: Untreated mouse 2: Lgr5+Lrig1+ population

city: Bruxelles

first: Adriana

last: Sanchez-Danes

address: Route de Lennik 808

country: Belgium

institute: Université Libre de Bruxelles

hyb_protocol: Affymetrix MG-430 PM array strips were hybridized with 8 mg of labeled cDNA, washed, stained and scanned according to the protocol described in GeneAtlas 3’IVT express kit User Manual.

scan_protocol: Arrays were scanned with GeneAtlasImaging station scanner (Affymetrix, Santa Clara, CA)

data_processing: Normalized expression signals were calculated from Affymetrix CEL files using RMA algorithm (Irizarry et al., 2003)

label: Biotin

source_name: Untreated_ Lgr5+Lrig1+ population

label_protocol: cDNA was labeled using GeneChip Mapping 250K Nsp Assay Kit (Affymetrix; catalog # 900766), according to manufacturer's instructions.

growth_protocol: Mouse colonies were maintained in a certified animal facility in accordance with the European guidelines.

extract_protocol: RNA isolation and cDNA amplification was performed as described in Gonzalez-Roca et al. (2010).

treatment_protocol: Sample preparation was performed as previously described in Jensen et al, Nature Protocols 2010.The back skin of mice bearing basal cell carcinomas and drug-tolerant lesions was placed in a petri dish and a sterile scalpel was used to remove the adipose tissue and muscle. The skin was incubated in thermolysin (Sigma) for 1h at 37oC and after a scalpel was used to separate epidermis from the dermis. The epidermal tissue was chopped in little pieces and resuspended in PBS supplemented with 5% chelated fetal calf serum and filtered in 70um and 40um cell strainers (BD). Cells were stained using the anti-Lrig1 (goat polyclonal, R&D) followed by the secondary  donkey anti-Goat-Alexa 647 ( Invitrogen). Living cells were selected by forward scatter, side scatter, doublets discrimination and by Hoechst dye exclusion. Tumour cell subpopulations were selected based on the expression of endogenous Lgr5-GFP and Lrig1-Alexa647.The cell sorting  was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences).

tag: genotype/variation

value: Untreated

value: Back skin

value: basal cell carcinoma tumour cells

tag: cell population


title: Vismodegib mouse 2: Lgr5+Lrig1+ population

source_name: Vismodegib_ Lgr5+Lrig1+ population

value: Vismodegib-treated


title: F-MDA5 Input, replicate2

description: Total RNA

value: inducible F-MDA5 BC-3 cells


title: F-MDA5 fRIP, replicate2

description: fRIP RNA


title: Vismodegib mouse 2: Lrg5-Lrig1+ population

source_name: Vismodegib_ Lrg5-Lrig1+ population


title: Vismodegib mouse 1: Lrg5-Lrig1+ population


title: Untreated mouse 2: Lrg5-Lrig1+population

source_name: Untreated_ Lrg5-Lrig1+population


title: Vismodegib mouse 1: Lgr5+Lrig1+ population


title: Untreated mouse 1: Lgr5+Lrig1+ population


title: F-RIG-I fRIP, replicate1

description: fRIP RNA


title: F-RIG-I Input, replicate2

description: Total RNA


title: F-MDA5 fRIP, replicate1

description: fRIP RNA


title: Huh7.5_BMP6_Replicate5

city: Oxford

first: Hal

last: Drakesmith

address: John Radcliffe Hospital

department: MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

description:
Huh7.5 cells treated for 24h with 18nMBMP6
SAMPLE 10

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were normalized using the Lumi package (Bioconductor) and compared using LIMMA (Bioconductor)

source_name: Huh7.5 cells, BMP6 18nM for 24h

label_protocol: Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays

growth_protocol: Huh7.5 cells were cultured in DMEM media supplemented with 10% FCS, 2mM glutamine, 100U/mL penicillin, 0.1mg/mL streptomycin prior to trypsinisation and plating in tissue culture plates

extract_protocol: Total RNA was isolated using Qiagen RNeasy mini spin columns, then converted into labelled cRNA and used for hybridisation; quality control was performed with Agilent Bioanalyser.

treatment_protocol: Cells were plated at 3x10e5/well in 12-well plates, allowed to adhere overnight, and were then treated for 24h with 18nM recombinant human BMP6 or media vehicle (untreated)


title: F-MDA5 Input, replicate1

description: Total RNA


title: Huh7.5_Untreated_Replicate6

description:
Untreated control
SAMPLE 12

source_name: Huh7.5 cells, untreated, 24h


title: Huh7.5_Untreated_Replicate4

description:
Untreated control
SAMPLE 8


title: Huh7.5_Untreated_Replicate5

description:
Untreated control
SAMPLE 9


title: Huh7.5_BMP6_Replicate6

description:
Huh7.5 cells treated for 24h with 18nMBMP6
SAMPLE 11