title: Lentivirus mediated CPEB1 overexpression in human foreskin fibroblasts-2 [RNA-Seq]

description:
CPEB1_OE2
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

data_processing:
RNA-seq data was aligned to the human hg19 genome build using Olego and Star aligners, and alternative splicing and alternative polyadenylation were estimated as described below.
Quantas software as described in Charizanis et al, 2012, Neuron, was used to estimate alternative splicing. Olego aligned alignment files were used to count observed junction reads for each exon. Weighted number of exon or exon-junction fragments uniquely supporting the inclusion or skipping isoform of each cassette exon and a probability score was assigned to each isoform. A Fisher’s exact test was used to evaluate the statistical significance of splicing changes using both exon and exon-junction fragments, followed by Benjamini multiple testing correction to estimate the false discovery rate (FDR). In addition, inclusion or exclusion junction reads were used to calculate the proportional change of exon inclusion (dI). See documentation at http://zhanglab.c2b2.columbia.edu/index.php/Quantas_Documentation.
Tandem UTR isoform analysis was performed with the MISO algorithm v0.5.2 using default settings as described in Katz et al, Nature, 2010, except for use of custom 3′UTR isoform annotations. We used a Bayes-factor threshold of 10,000 and difference values (delta Psi) with an absolute value of at least 0.03
For TAIL-seq data analysis, image files were downloaded from the MiSeq and run on tailseeker (Chang et al, 2014, Mol Cell) to determine accurate polyA tail lengths, yielding paired fastq files corresponding to the 5' (R5) and 3' (R3 polyA tail) ends of each read. Reads were aligned against the human genome (hg19) and viral genomes (Human_Herpesvirus_5_strain_Merlin, AY446894.2) using STAR under default parameters. Features were assigned using Subread with gencode v19 annotations and with Human_Herpesvirus_5_strain_Merlin features, and filtered to obtain only the uniquely mapped protein coding genes. For analysis of virally mapped reads, all genes were counted. Reads with tails measuring 0 lengths were removed. For genes with at least 20 mapped reads, median lengths were measured and the global distributions of these lengths were compared against each other using the Kolmogorov-Smirnov test. For each gene captured in all samples and with at least 20 mapped reads, individual tail length distributions were compared amongst samples using the Mann Whitney U test with a P value cutoff of 0.025
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph
Supplementary_files_format_and_content: TableS3: Significantly changing alternative host polyadenylation events
Supplementary_files_format_and_content: TableS6: Significant host gene alternative cassette splicing changes upon CPEB1 KD and CPEB1 OE from RNA-seq
Supplementary_files_format_and_content: TableS8: PolyA tail lengths analyzed by TAIL-seq

growth_protocol: Human Foreskin Fibroblasts were grown in MEM supplemented with 10% fetal bovine serum

extract_protocol:
Total RNA was isolated using TRIzol reagent using the manufacturer’s protocol (Life Technologies)
For RNA-seq, libraries were made using the Illumina polyA selection truseq library kit using different barcodes. Libraries were not strand specific. TAIL-seq was performed as described by Chang et al, (2014, Molecular Cell), with minor modifications. Briefly, total RNA was extracted from cells by TRIzol reagent (Invitrogen, 15596-018) according to the manufacturer’s protocol. The RNA ligated to 3′ “splint” oligonulceotide (NNNGTCAGTTTTTTTTT) to enrich for polyadenylated transcripts and partially digested by RNase T1 (Ambion, AM2283). The fragmented RNAs were pulled down with Dynabeads M-280 Streptavidin (Invitrogen, 11206D) magnetic beads, phosphorylated on bead using T4-PNK reaction (NEB), eluted using 2X RNA loading dye (with 95% formamide), run on a 6% polyacrylamide urea gel (NuPage), stained with SYBR gold (Thermo) and gel purified in the range of 250 - 750 nucleotides. The purified RNAs were ligated to 5′ adapter (Illumina Truseq small RNA kit), reverse transcribed using Superscript III (Invitrogen) and amplified by PCR using Phusion DNA polymerase (Thermo, F-530L) and Illumina Truseq small RNA universal forward and indexed (barcode) reverse primer(s). The library was purified using AMPureXP beads (Agilent) and the libraries were sequenced on an Illumina Miseq instrument (51 ~ 251 paired end run) with 10% of the PhiX control library (Illumina, FC-110-3001) and 1% of the spike-ins mixture of known polyA-containing DNAs
Non-strand specific. TAIL-seq using Miseq

treatment_protocol:
CPEB1, UL99 and control GFP lentiviruses were generated in 293T cells with PEI transfection reagent, in 10 cm format with 107 cells per dish, using the second generation packaging constructs psPAX2 and pMD2.G. Supernatants were harvested at 60 h post-transfection and 0.22 µm filtered. Freshly seeded HFFs were transduced at an MOI of 0.5 to 1, without polybrene or additional reagents. Puromycin selection was initiated in the transduced HFFs at 48 h, and cells were harvested following 3 days of drug selection.
HFFs were either mock-treated or treated with siCPEB1 (Dharamcon) or a Non-targeting siRNA control 24 h post-seeding following the manufacturer's protocol and media was replaced by fresh HFF growth media 24 h after transfection.

tag: treatment

value: Puromycin selection - 5 days


title: NPC_Mock_48h

status: Public on Sep 17 2016

type: SRA

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

country: USA

institute: UCSD

description: Sequencing Sample 7

library_source: transcriptomic

data_processing:
RNA-seq raw reads were trimmed for adaptor sequences or low-quality bases and mapped to both the human genome (hg19 build) and the HCMV Merlin genome (Genbank AY446894.2) with GSNAP, with additional filtering for repetitive regions. Virus-mapping reads were obtained from the HSV-2 and HIV-1 samples using Genbank accessions NC_001798.1 and K03455.1, respectively.
Genome coverage files for visualization (bigWig format) were generated using UCSC tools bedGraphToBigWig and genomeCoverageBed, and strand-corrected.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig (each .bw includes human transcriptome only)

molecule: polyA RNA

organism: Homo sapiens

growth_protocol: HFFs were cultured as described in Sanders et al. 2008; ECs as in DuRose et al., 2012; and ES-derived NPCs as in Belzile et al., 2014.

extract_protocol:
All HCMV and HSV-2 materials were collected at the time of harvest by trypsinization and centrifugation, and snap-frozen prior to RNA extraction. HIV-1 materials were harvested directly into TRIzol reagent. TRIzol RNA extractions of RNA were performed according to the manufacturer’s instructions (Invitrogen/Life Technologies).
All HCMV and HIV-1 sample conditions were subjected to strand-specific RNA-seq using the dUTP method (Parkhomchuk et al., 2009) with adaptations as described previously (Huelga et al., 2012). HSV-2 libraries were prepared with Illumina TruSeq Stranded mRNA Sample Preparation reagents.
Strand-specific RNA-seq

treatment_protocol: HCMV infections, all with strain TB40E, were performed at an MOI of 5, as described in Belzile et al., 2014. HUES9 ES cells used to derive the NPCs. HSV-2 infections were performed in HFFs with strain G at an MOI of 10, including an initial 30 min adsorption period at 4°C. HIV-1 infections of IL-2-activated CD4+ T-cells were performed as described in Noraz et al., 1997.

tag: cell type

value: NPC cells (ES-derived)

tag: infection

value: mock

tag: time


title: NPC_InfectedHCMVTB40E_48h

description: Sequencing Sample 8

data_processing:
RNA-seq raw reads were trimmed for adaptor sequences or low-quality bases and mapped to both the human genome (hg19 build) and the HCMV Merlin genome (Genbank AY446894.2) with GSNAP, with additional filtering for repetitive regions. Virus-mapping reads were obtained from the HSV-2 and HIV-1 samples using Genbank accessions NC_001798.1 and K03455.1, respectively.
Genome coverage files for visualization (bigWig format) were generated using UCSC tools bedGraphToBigWig and genomeCoverageBed, and strand-corrected.
For HCMV mRNA abundance measurements, a secondary iteration of mapping was performed against the TB40E LISA genome (Genbank KF297339.1) using Bowtie. Resulting unique read mappings from the HCMV-infected datasets (bed format) are listed with raw read totals provided as terminal values in the read IDs.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig (each .bw includes human transcriptome only)
Supplementary_files_format_and_content: bed (each .BED includes HCMV mRNA abundance measurements)

value: HCMV (strain TB40E)


title: E12.5 [86]

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

country: Germany

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

description: Sample 20

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using TopHat with default parameters.
mRNA quantification according to RefSeq Transcripts - 2014-01-03.
raw read counts were normalized by DE-Seq.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include DE-Seq normailzed expression values for each Sample.

molecule: total RNA

organism: Mus musculus

source_name: DRG neuron

growth_protocol: Adult lumbar mouse DRG were dissected, dissociated and plated at low density on laminin-coated (5 μg/ml, Roche) dishes. Neurons were cultured in Neurobasal-A medium supplemented with B-27 (Gibco), and maintained in a humidified atmosphere of 5% CO2 in air at 36.5°C.

extract_protocol:
Cells were isolated by cell sorting, lyzed in Qiazol and RNA isolated. Illumina TruSeq RNA Sample Prep Kits V2 was used with 10 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: developmental stage

value: embryonic 12.5

tag: time after plating


title: Lentivirus mediated CPEB1 overexpression in human foreskin fibroblasts-1 [RNA-Seq]

description:
CPEB1_OE1
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

value: HFF cells

tag: genotype/variation


title: EC_InfectedHCMVTB40E_48h

description: Sequencing Sample 5

value: EC cells


title: E12.5 [84]

description: Sample 19


title: Lentivirus mediated GFP overexpression in human foreskin fibroblasts [RNA-Seq]

description:
GFP_OE
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

value: Lentivirus mediated GFP overexpression


title: Sham operated [70]

description: Sample 17

value: adult


title: DSN_NB4_2392_48h_rnaseq

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

description: RNA-seq

data_processing:
Basecalls performed using CASAVA
Reads were aligned to the hg18 genome assembly using ELAND
Peaks were called using MACS 1.3.3 on settings:mfold =32, tsize=32, p value = e-6
Genome_build: All ChIP-seq tracks were mapped to genome build HG18, RNA-seq tracks were mapped to HG18.
Supplementary_files_format_and_content: wig files were generated (extended to 300bps)

source_name: MC2392 treated NB4 cells, 48h  RNA

growth_protocol: NB4 cells were grown at 37°C in air and 5% CO2 in RPMI 1640 medium (EUROCLONE),supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma),  ampicillin/streptomycin and 0.1% gentamicin (SIGMA).1% L-glutamine, 1%

extract_protocol:
Chromatin was sonicated and used for ChIP-seq using H3K9K14ac (Diagenode). RNA was extracted using qiagen RNeasy kit according to manufacturer. cDNA first and Ds-cDNA synthesis then, was  performed following supplier’s instructions (Invitrogen). Ds-cDNA samples were prepared for  sequencing by end repair of 20 ng DNA as measured by Qubit (Invitrogen). Adaptors were ligated to DNA fragments, followed by size selection (~300 bp) and limited PCR amplification (14 cycles). The resulting sample libraries were subjected to DSN treatment (Evrogen) according to the manufacturer’s recommendations.
ChIP and RNA seq libraries were preapared according to  Illumina's standard protocol.

treatment_protocol: NB4 cells were treated with ATRA, MS275 and MC2392

value: NB4 promyelocytic leukaemia cell line

tag: reference genome


title: ChIP:H3K27ac_ab4729_KOPT-K1_052616 [lab: Look-DFCI]

city: Cambridge

first: Richard

middle: A

last: Young

state: MA

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

library_source: genomic

data_processing:
Aligned using /usr/local/bin/bowtie with configuration -p 4 --best -k 2 -m 2 --sam -l 40
hg19
Supplementary_files_format_and_content: WIG files(s) represent counts of aligned reads within 50 bp bins with each read being extended 200 in the direction of alignment. Counts are in reads-per-million and floored at 0.1

molecule: genomic DNA

source_name: T-ALL

growth_protocol: Human KOPTK1 cells were purchased from the American Type Culture Collection (ATCC), Manassas, VA. Cells were maintained under typical conditions in RPMI media with 10% Bovine Calf Serum. For location analysis, cells were grown to a density of 1 million per ml and 99% viability prior to cross-linked with formaldehyde for 20 min.

extract_protocol:
Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.
Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies.

tag: cell line

tag: tissue source

value: Blood

tag: chip antibody

tag: antibody manu.

value: Abcam

tag: antibody catalog


title: HCMV infected siCPEB1 siRNA treated human foreskin fibroblasts [RNA-Seq]

description:
siCPEB1
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

value: HCMV infected

value: CPEB1 siRNA treatment


title: EC_InfectedHCMVTB40E_96h

description: Sequencing Sample 6


title: Sham operated [69]

description: Sample 16


title: HFF_InfectedHCMVTB40E_96h

description: Sequencing Sample 3


title: DSN_NB4_2392_24hrnaseq

description: RNA-seq

source_name: MC2392 treated NB4 cells, 24h RNA


title: Sham operated [123]

description: Sample 18


title: DSN_NB4Atra24hrnaseq

description: RNA-seq

source_name: ATRA treated NB4 cells, 24h RNA

value: ATRA 24h


title: LID84

city: Bundoora

first: Nina

last: Riddell

state: Victoria

address: La Trobe University, Plenty Rd.

department: Psychology and Public Health

country: Australia

institute: La Trobe University

data_processing:
Read quality was assessed using FastQC
Adapter and low quality (Q-score <10) sequences removed using CutAdapt and Trimmomatic
Reads were mapped to the chick genome using Tophat2 and Bowtie2
The number of reads uniquely mapping to each gene was counted using existing gene models with HTSeq
Genome_build: GalGal4
Supplementary_files_format_and_content: The txt file was generated using the data processing steps described above. It is matrix table containing processed data for all samples (raw counts)

organism: Gallus gallus

source_name: Retina, RPE, choroid

extract_protocol:
Total RNA was isolated from the retina/RPE/choroid using the miRNeasy Mini Kit (Qiagen) including DNase digestion. RNA quality and quantity was assessed on the 2100 Bioanalyzer. All samples had an RNA integrity number (RIN) of >8.3. RNA quantity was also assessed on the Qubit 2.0 Fluorometer.
Libraries were prepared using the TruSeq Stranded mRNA LS kit (Illumina) with dual indexing according to the manufacturer’s instructions. The generated libraries were assessed on the 2100 Bioanalyzer to ensure an average size distribution of approximately 280bps, then quantified on the Qubit 2.0 Fluorometer and by qPCR. Libraries were normalised to 10nM in Tris-HCl, pooled, and prepared for cluster generation on the Illumina cBot using the TruSeq SR Cluster Kit V3-cBot (Illumina) with denatured template DNA diluted to 7pM. The flow cell and sequencing reagents (TruSeq SBS Kit V3; Illumina) were loaded on the Illumina HiSeq 1500 and a dual-index, single-end, 100bp sequencing run performed.

treatment_protocol: Male chicks (Leghorn/New Hampshire) were raised from post-hatch days 0-4 under a 12-hr day/night light cycle in groups of <25.  In the middle of the light cycle on day 5, chicks were randomly assigned to a lens condition (+10 or -10 dioptres, or No Lens) and lenses (Polymethyl Methacrylate) attached to Velcro were fixed to the periocular feathers of the right eye. Following a further 1, 2, or 3 days with lenses attached, chicks were anaesthetized and right eye refraction and axial dimensions determined by retinoscopy and A-Scan ultrasonography. Chicks were euthanized and their right eyes were enucleated. The retina/RPE/choroid was immediately collected from the posterior eyecup and frozen in liquid nitrogen before being transferred to -80°C.

tag: tissue

value: Retina, RPE, choroid

tag: Sex

value: male

tag: breed

value: Leghorn/New Hampshire

tag: age

value: Post-hatch day 7

tag: time-point

value: 2 days (post-lensing)

tag: lens type

value: No Lens


title: EC_Mock_48h

description: Sequencing Sample 4

tag: cell type

tag: infection

value: mock


title: LID85


title: DSN_NB4_AtRA_48h_rnaseq

description: RNA-seq

source_name: ATRA treated NB4 cells, 48h RNA

tag: treatment

value: ATRA 48h


title: DSN_NB4_ctr_rnaseq

description: RNA-seq

source_name: DMSO NB4 cells RNA

value: DMSO


title: Peripheral lesion [75]

description: Sample 14

tag: developmental stage

tag: time after plating


title: Peripheral lesion [124]

description: Sample 15


title: HCMV infected Non-targeting siRNA treated human foreskin fibroblasts [RNA-Seq]

description:
HCMV
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

value: HFF cells

value: Non-Targeting siRNA treatment


title: HFF_Mock_48h

description: Sequencing Sample 1

tag: time


title: IsoG_Rep1

city: boston

first: jesse

middle: M

last: gray

address: 77 avenue louis pasteur, NRB 356

department: Genetics

institute: Harvard Medical School

description: Net-Seq

data_processing:
fastq trimming with fastx v0.0.13
alignment with STAR v2.2.1
mapped to RefSeq with bedtools v2.25.0
TSS and PAS centered count files generated with metaseq v0.5.6
genes of length greather than 10kb or 250kb were filted with different bed files.
genome build: hg19
Supplementary_files_format_and_content: Read counts by gene (rows) and TSS or PAS distance (columns). Each row is a gene, and each column is a 200 bp bin at a position relative to the TSS or PAS, with the first half of columns upstream and the 2nd half downstream of the TSS/PAS. (This is upstream and downstream are in genomic coordinates, not corrected for gene orientation.) For TSS_500arrays, each column is a 50bp bin around the TSS. In the filenames, F denotes reads aligning to the negative genomic strand, R denotes reads aligning to the positive genomic strand, Pos includes only annotated genes on the positive genomic strand, and Neg includes only annotated genes on the negative genomic strand.

source_name: Hela S3 cells

growth_protocol: Hela cells were cultured in DMEM and plated in 10cm plates the day prior to drug treatment.

extract_protocol:
Cells were washed and harvested using Qiagen RNeasy mini kit. 10ug of RNA was DNAse treated and cleaned up with Rneasy MinElute Cleanup Kit.
Total stranded RNA libraries were construced with a standard dUTP protocol.  For a the drug screen the previously published Tag-Seq protocol was followed.

treatment_protocol: Drugs were added to cells and DMSO was added at the same amount for the control.

value: Isoginkgetin

tag: dose

value: 30 uM

value: 6 hours

tag: biological replicate

tag: experiment number

value: Exp 6


title: LID77

value: Post-hatch day 6

value: 1 day (post-lensing)


title: HFF_InfectedHCMVTB40E_48h

description: Sequencing Sample 2

value: HCMV (strain TB40E)


title: DMSO_Rep2

description: Net-Seq


title: DMSO_Rep1

description: Net-Seq


title: 36h [349]

description: Sample 11

value: adult

value: 36 hrs


title: Peripheral lesion [74]

description: Sample 13


title: 36h [350]

description: Sample 12


title: LID74

value: Retina, RPE, choroid

value: male

value: Leghorn/New Hampshire


title: Mock infected Non-targeting siRNA treated human foreskin fibroblasts [RNA-Seq]

description:
Mock
Table S6 AS_Changes_siCPEB1_CPEB1OE_HFFs_RNA-seq.xlsx
Table S3 Host_APA_events.xlsx

value: Mock Infected (serum with no HCMV)


title: Rep2_barcode_RTPCR1

city: Barcelona

first: Guillaume

last: Filion

address: Dr. Aiguader 88

department: GRSCC

country: Spain

institute: CRG

description:
BHIVe transcripts with barcode, bio rep 2, tech rep 1
Jurkat_BHIVE_mini_expr.txt

data_processing:
Library strategy: B-HIVE
Sequencing lane demultiplexing: Sequences were demultiplexed using bcl2fastq. In case the index was inside the sequenced amplicon, demultiplexing was carried out with custom scripts. The used indices are specified in the last row of the Sample section.
Barcode extraction: Barcodes were extracted from the forward reads. The barcode sequences in the construct are flanked by the Illumina adaptor and the T7 promoter sequence (TATAGTGAGTCGTATTAAAA). The promoter sequence was identified using Seeq (http://github.com/ezorita/seeq) allowing up to 2 mismatches.
Barcode clustering: Barcodes were clustered by sequence similarity using starcode and allowing up to 1 mismatch (http://github.com/gui11aume/starcode).
Alignment: Reverse reads were aligned on GRCh37/h19 using BWA-mem with default parameters.
Filtering on alignment quality: Only SAM mapping qualities greater or equal than 20 were used in the analysis.
Filtering of recombinant molecules: A barcode was associated to a mapped locus based on their co-ocurrence in paired-end reads. Associations between a barcode and a mapped locus reported by less than 10 paired-end reads were discarded. The remaining associations were considered reliable if both the barcode and the mapped locus were found together at least 90% of the time.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: tab delimited tables
Supplementary_files_format_and_content: Jurkat_BHIVE_mini_integ.txt: BHIVE integration sites
Supplementary_files_format_and_content: Jurkat_BHIVE_mini_expr.txt: BHIVE transcript expression
Supplementary_files_format_and_content: Jurkat_BHIVE_mini_latent.txt: Latent integrations reference table
Supplementary_files_format_and_content: Jurkat_BHIVE_mini_drug.txt: Barcode reactivation upon treatment

source_name: Immortalized human T lymphocyte Jurkat cell line

growth_protocol: Jurkat human T cells line were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% Pen Strep and 1% GlutaMAX (100x) at 37℃ under a 95% air/5% CO2 atmosphere. Jurkat T cells were passaged every 2 days with 1 to 5 dilution. After FACS sorting 4 days post infection, 20,000 GFP-positive cells were maintained in the same medium for the next 17 days without passage.

extract_protocol:
Genomic DNA and total RNA from the same infected cell pool were extracted by the AllPrep DNA/RNA Mini Kit. Total RNA was further proceeded to purify mRNA by Oligotex mRNA Mini Kit.
Libraries used for mapping HIV integrations were prepared by inverse PCR. Briefly, 3 µg DNA were harvested and digested by the restriction enzyme BplI. Fragments were blunt-end self-ligated in the total volume 1 mL at 16℃ overnight. The pellet was dissolved in 84 µL distilled water for plasmid-safe DNase treatment. DNase-treated samples were cleaned up by the QIAquick PCR purification kit. Samples were eluted in 20 µL EB buffer. 8 µL eluted product were used for two rounds of nested PCR. PCR products with different indexes were pooled together in the final concentration of 4 nM for sequencing 50 bp paired end on a Illumina NextSeq sequencer. RNA and DNA libraries were prepared by RT-PCR and regular PCR, respectively, with the same primers. RNA and DNA libraries were sequenced 50 bp single read by on a Illumina NextSeq sequencer.

treatment_protocol: Samples used for the latency reversal experiments were treated with the indicated drugs for 24 hours before performing DNA and RNA extraction.

value: T lymphocyte

tag: days of post infection for sample analysis

tag: drug treatment

tag: demultiplex indices used

value: CGTA, GATC, TCAG, TCGA


title: LID82

tag: tissue

tag: Sex

value: Post-hatch day 7

value: 2 days (post-lensing)

value: No Lens


title: Rep1_barcode_RTPCR1

description:
BHIVe transcripts with barcode, bio rep 1, tech rep 1
Jurkat_BHIVE_mini_expr.txt

value: GATC, GCTA, ATCG, ATGC


title: LID68

tag: breed

tag: age

value: Post-hatch day 8

value: 3 days (post-lensing)

value: Negative (10 diopter)


title: 24h [346]

description: Sample 8

value: 24 hrs


title: LID60

tag: time-point

tag: lens type


title: Rep1_barcode_RTPCR2

description:
BHIVe transcripts with barcode, bio rep 1, tech rep 2
Jurkat_BHIVE_mini_expr.txt

tag: cell type

value: AGCT, TGCA, AGTC, TACG


title: LID73

value: Post-hatch day 6

value: 1 day (post-lensing)


title: 36h [348]

description: Sample 10

value: adult

value: 36 hrs


title: Rep2_barcode_RTPCR2

description:
BHIVe transcripts with barcode, bio rep 2, tech rep 2
Jurkat_BHIVE_mini_expr.txt

value: ACTG, GTCA, CTGA, TGAC


title: 24h [347]

description: Sample 9


title: LID69

value: Retina, RPE, choroid

value: male

value: Leghorn/New Hampshire


title: 06h [341]

description: Sample 3

value: 6 hrs


title: LID48

value: Post-hatch day 7

value: 2 days (post-lensing)


title: 12h [344]

description: Sample 6

value: 12 hrs


title: IsoG_Rep2

description: Net-Seq

tag: treatment

value: Isoginkgetin

value: 30 uM

value: 6 hours

value: Exp 6


title: 24h [345]

description: Sample 7

tag: developmental stage

value: 24 hrs


title: LID59

value: Post-hatch day 8

value: 3 days (post-lensing)

value: Negative (10 diopter)


title: LID8

value: Post-hatch day 6

value: 1 day (post-lensing)

value: Positive (10 diopter)


title: Rep2_barcode_PCR2

description:
BHIVe integrated barcodes, bio rep 2, tech rep 2
Jurkat_BHIVE_mini_expr.txt

value: T lymphocyte

value: GCAT, CTAG


title: Rep2_integration_map

description:
Map HIV integraion sites in the second replicate
Jurkat_BHIVE_mini_integ.txt


title: 12h [343]

description: Sample 5

value: adult

tag: time after plating


title: LID58

value: Retina, RPE, choroid

value: male

value: Leghorn/New Hampshire

value: Post-hatch day 7


title: Rep1_integration_map

description:
Map HIV integraion sites in the first replicate
Jurkat_BHIVE_mini_integ.txt


title: 12h [342]

description: Sample 4

value: 12 hrs


title: Rep2_barcode_PCR1

description:
BHIVe integrated barcodes, bio rep 2, tech rep 1
Jurkat_BHIVE_mini_expr.txt

value: CAGT, CGAT


title: Rep1_barcode_PCR2

description:
BHIVe integrated barcodes, bio rep 1, tech rep 2
Jurkat_BHIVE_mini_expr.txt

value: GTAC, TAGC


title: Day 20 conceptus_D20-3

city: Columbia

first: Thomas

last: Spencer

state: MO

address: 920 East Campus Drive

department: Animal Sciences

institute: University of Missouri

description: Sample name: D20-3

data_processing:
Analysis of RNA-seq data was completed using a CLC Genomics Server 6.0 and CLC Genomics Workbench 7.0.4 (CLC bio, Aarhus, Denmark).
Ion reads were quality trimmed (error probability 0.02) with default parameters.
Illumina reads were quality trimmed (error probability 0.001) with default parameters and 13 bp were removed from the 5’ end of each read.
All sequences were mapped to the Ovis aries genome (Oar_v3.1) with Ensembl version 75 annotations requiring paired mapping and using reads per kilobase of transcript per million (RPKM) for expression values.
Genome_build: Ovis aries genome (Oar_v3.1) with Ensembl version 75 annotations
Supplementary_files_format_and_content: Processed file includes expression values (RPKM) for each sample with gene symbol and description from Ensembl 75. The RawData tab includes all genes and the RPKM5 tab includes only genes with RPKM ≥ 5 in all samples

organism: Ovis aries

source_name: conceptus

growth_protocol: In utero conceptus development

extract_protocol:
Uteri frozen in OCT were cryosectioned (10 µm) using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). Three tissue sections were mounted per slide to RNase-free polyethylene naphthalate-coated slides (Carl Zeiss, Munich, Germany) and immediately placed on dry ice. Slides were fixed and stained immediately following sectioning using previously described methods. Briefly, slides were transferred from dry ice into ice-cold 95% ethanol for 30 sec, then washed in 75% ethanol for 30 sec. Tissue sections were stained in freshly filtered 1% cresyl violet solution in 75% ethanol for 1 min. Tissue sections were then dehydrated through 75% ethanol (30 sec) and 95% ethanol (30 sec), followed by two 30 sec and one 5 min incubation in anhydrous 100% ethanol. Slides were dried for 10 min at room temperature and stored in sealed containers at -80ºC until use. The LE and GE were visually identified and separately captured from a single slide of three uterine sections for each animal. Slides were discarded after 60 min of use on the laser capture microdissection (LCM) LMD7 microscope (Leica Microsystems). Total RNA was extracted from cells collected from the LE and GE using the RNeasy MinElute kit (Qiagen, Valencia, CA). Prior to sequencing, 5 ng of total RNA was amplified using the Ovation RNA-Seq System V2 (cat. # 7102; Nugen, San Carlos, CA). The resulting single primer isothermal amplified (SPIA) cDNA was cleaned using the QIAquick PCR Purification Kit (Qiagen). Day 12 conceptuses: Total RNA was extracted from day 12 conceptuses (n = 5) using the RNeasy plus kit with on column DNase digestion (Qiagen). Total RNA quality and quantity was determined using a RNA 6000 Pico Kit and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). A total of 374-512 ng of RNA was collected from each conceptus, for each animal. Prior to sequencing, 50 ng of total RNA was amplified using the Ovation RNA-Seq System V2 (Nugen). Days 14, 16 and 20 conceptuses: Total RNA was extracted from conceptuses (n = 5 per day) and quality checked as described for day 12 conceptuses. RNA samples were enriched for mRNA by selective depletion of ribosomal RNA using RiboMinus Eukaryote Kit for RNA-Seq (cat. # A1083708, Thermo Fisher Scientific Inc., Wilmington, DE).
LE and GE: The Ion Plus Fragment library kit (Life Technologies, Waltham, MA) was used to construct Ion Proton sequencing libraries from SPIA LCM cDNA (200 ng). Sonication (Bioruptor, Diagenode Inc., Denville, NJ) was used to fragment the cDNA. End repaired cDNA was purified using 1.0X v/v AMPure XP beads (Beckman-Coulter Genomics, Danvers, MA). Following barcode adapter ligation and 7 cycles of amplification, the final libraries were size selected by capturing the fragments bound between 0.7X and 1.0X v/v AMPure XP beads. Emulsion PCR was performed on an Ion Chef instrument using the Ion P1 Hi-Q reagents. Sequencing beads were quantified and evaluated by flow cytometry using a Guava Easy Cyte (Millipore, Billerica, MA) with Sybr Gold (Molecular Bioprobes, Eugene, OR) before loading on an Ion P1 v3 semiconductor sequencing chip. Libraries were sequenced on an Ion Proton using Ion P1 Hi-Q sequencing reagents and 520 flow cells (Life Technologies) by the Washington State University Molecular Biology and Genomics Core. Three barcoded libraries were pooled per P1 chip for sequencing. Base calling and primary analysis was performed using Torrent Suite 4.6. Day 12 conceptus: Following amplification, libraries were constructed and sequenced at the University of Missouri DNA Core following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq DNA PCR-Free sample preparation kit (cat. # FC-121-3001). Briefly, cDNA (0.5 µg) was sheared using standard Covaris methods (Covaris, Inc., Woburn, MA) to generate average fragmented sizes of 350 bp. The resulting 3’ and 5’ overhangs were converted to blunt ends by an end repair reaction which uses a 3’ to 5’ exonuclease activity and polymerase activity. A single adenosine nucleotide was added to the 3’ end of the blunt fragments followed by the ligation of Illumina indexed paired-end adapters. The adaptor ligated library was purified twice with sample purification beads and quantified using the KAPA library quantification kit (cat. # KK4824; Kapa Biosystems, Inc., Wilmington, MA) and library fragment size confirmed on the Fragment Analyzer (cat. # DNF-472; Advanced Analytical Technologies, Inc., Ankeny, IA). Libraries were diluted and sequenced as 100 bp paired-end sequences on the Illumina HiSeq 2500 platform. Day 14, 16 and 20 conceptus:Libraries were prepared and sequenced by Global Biologics LLC (Columbia, MO, USA). Ribosome-depleted RNA was used as input for the Illumina TruSeq Stranded mRNA HT library construction procedure (San Diego, CA, USA). Prior to sequencing, RNA was fragmented and RT primers were hybridized for cDNA synthesis. The resulting cDNA was prepared for sequencing by 3’ adenylation, adapter ligation, and PCR amplification (98ºC for 30 sec; 14 cycles of 98ºC for 10 sec, 60ºC for 30 sec, 72ºC for 30 sec; 72ºC for 5 minutes). Library validation was performed using a Fragment Analyzer (Advanced Analytical) with HiSens NGS reagents followed by quantitation using the Qubit HS DNA assay (cat. # Q32851; Thermo Fisher Scientific, Inc.) and qPCR library kit for Illumina platforms (Kappa Biosystems, Inc.). Libraries were pooled and sequenced as 100 bp paired-end sequences on the Illumina HiSeq 2500 platform.

treatment_protocol: Conceptuses were flushed from the uterine horn with 20 ml of 10 mM PBS (pH 7.2). Conceptus tissue was transferred to a 1.5ml microcentrifuge tube and frozen in liquid nitrogen. Several sections (~0.5 cm) from the mid-portion of each uterine horn ipsilateral to the ovary were placed in Optimum Cutting Temperature Compound (OCT), frozen in liquid nitrogen vapor, and stored at -80°C.

value: conceptus

tag: day of pregnancy

tag: isolation method

value: whole tissue

tag: rna processing

value: Ribosome depleted RNA


title: LID57

value: 2 days (post-lensing)

value: Negative (10 diopter)


title: LID49


title: Rep1_barcode_PCR1

description:
BHIVe integrated barcodes, bio rep 1, tech rep 1
Jurkat_BHIVE_mini_expr.txt

value: ACGT, CATG


title: 06h [340]

description: Sample 2

value: 6 hrs


title: 06h [339]

description: Sample 1


title: LID46

value: Post-hatch day 6

value: 1 day (post-lensing)


title: LID45


title: LT2, Baseline LB rep3

city: Wan-Ju-Goon

first: Sanghyun

last: Han

address: 166 Nong-Saeng-Myung-Ro

country: South Korea

institute: RDA-NAAS

data_processing:
Illumina Casava v1.8.2 was used for basecalling.
Sequenced reads were trimmed of adaptor and barcode sequences using Trimmomatic (Bolger et al., 2014) with parameters ILLUMINACLIP:<adaptor.fasta>:2:30:10 HEADCROP:10 SLIDINGWINDOW:4:20 MINLEN:70, then mapped to S. Typhimurium LT2 whole genome (NC_003197 and NC_003277) using bowtie v2.1.0 and tophat v2.0.9 with tophat parameters --library-type=fr-firststrand.
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using a protocol from Trapnell et al., Nature Protocols, 2012.  In short, the reads and the merged assembly were fed to Cuffdiff which normalizes read counts into FPKM (fragments per kilobase of transcript per million mapped fragments), calculates expression levels, and tests the statistical significance of observed changes in expression levels.
Genome_build: NC_003197 and NC_003277
Supplementary_files_format_and_content: Tab-delimited files including averaged FPKM values for each condition.

organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

source_name: Bacterial cells grown on LB

growth_protocol: Salmonella cells were grown on either shoots or roots of tomato plants for 3 days until the cells were retrieved from the plants for mRNA isolation.

extract_protocol:
Tomato plants maintaining Salmonella cells were submerged in Qiagen RNAprotect solution to stabilize bacterial RNA.  Total RNA was isolated from the bacterial cells dislodged from the plants using Qiagen RNeasy mini kit.  The total RNA was depleted of prokaryotic ribosomal RNA using Epicentre Ribo-Zero rRNA removal kit.
RNA libraries were prepared for sequencing using standard Illumina protocols.

tag: strain

tag: growth media

value: LB


title: LT2, Baseline LB rep2


title: Day 20 conceptus_D20-5

description: Sample name: D20-5


title: LID44

value: Retina, RPE, choroid


title: Day 16 conceptus_D16-5

description: Sample name: D16-5


title: LID36

value: male

value: Post-hatch day 8

value: 3 days (post-lensing)

value: Positive (10 diopter)


title: Day 20 conceptus_D20-1

description: Sample name: D20-1


title: LID26

value: Leghorn/New Hampshire


title: LID39


title: Day 16 conceptus_D16-4

description: Sample name: D16-4


title: LID33


title: Day 20 conceptus_D20-4

description: Sample name: D20-4


title: LID32


title: LID2


title: LT2, Baseline LB rep1


title: dnaj1 KD 2

city: Kansas City

first: Madelaine

last: Gogol

address: 1000 E. 50th Street

department: Computational Biology Core

institute: Stowers Institute

description:
dnaj_kd_2
PolyA RNA

data_processing:
Base-calling and bcl to fastq generation was done with CASAVA 1.8.2 from Illumina.
RNA-seq was aligned to genome dm3 from UCSC using tophat 2.0.10 with arguments --solexa-quals --segment-mismatches 1 -x 1 -g 1 --no-coverage-search. ChIP-seq was aligned to dm3 using bowtie2 2.1.0 with -k 1.
bigWig tracks were generated in R 3.0.2 by dividing by the total number of alignments and multiplying by 1e6 (rpm).
Peaks were called using macs2 (2.0.10.20120913).
Differentially expressed genes were called with edgeR 3.4.2.
Genome_build: dm3 (Release 5)
Supplementary_files_format_and_content: bigWig tracks with rpm-normalized coverages.

organism: Drosophila melanogaster

source_name: Drosophila S2 cells, dnaj1 KD

growth_protocol: Drosophila S2 cells were grown in Schneider's medium supplemented with 10% fetal bovine serum.

extract_protocol:
Total RNA was isolated from S2 cells treated with either dsRNA against lacZ, mlf, bag2, or dnaj-1 using TRIzol (Invitrogen). Two separate biological experiments were performed for each RNA-seq analysis.
RNA-seq libraries were prepared using TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer's protocol.

treatment_protocol: Drosophila S2 cells were grown in Schneider's medium supplemented with 10% fetal bovine serum to a density of 3-6 x10^6 cells/ml. Cells were diluted to 1x10^6 cells/ml in serum-free media and incubated with 10 μg dsRNA per 1 x10^6 cells for 45 min at 25°C. An equal volume of Schneider's media containing 10% serum was added to the cells and the cells were grown for 4 days at 25°C before collection for ChIP, RNA, and/or protein isolation. dsRNA against lacZ was used as a control for all RNAi experiments.

tag: cell line

tag: genotype/variation

value: dnaj1 KD


title: LT2, Tomato Root rep1

source_name: Bacterial cells grown on Tomato root

value: tomato root


title: LT2, Tomato Root rep3


title: LT2, Tomato Root rep2


title: Day 16 conceptus_D16-1

description: Sample name: D16-1

tag: tissue

value: conceptus

value: whole tissue


title: LID18

tag: Sex

value: Post-hatch day 7

value: 2 days (post-lensing)


title: LID16

tag: breed


title: Day 14 conceptus_D14-4

description: Sample name: D14-4

value: Ribosome depleted RNA


title: LT2, Tomato Shoot rep3

source_name: Bacterial cells grown on Tomato shoot

value: tomato shoot


title: Day 14 conceptus_D14-3

description: Sample name: D14-3


title: LT2, Tomato Shoot rep2


title: Day 12 conceptus_D12-4

description: Sample name: D12-4


title: LID15

tag: age


title: Day 14 conceptus_D14-2

description: Sample name: D14-2


title: LID13

tag: time-point


title: Day 12 conceptus_D12-2

description: Sample name: D12-2


title: LT2, Tomato Shoot rep1


title: HCMV infected Non-targeting siRNA treated human foreskin fibroblasts [Tail-Seq]

description:
HCMV_infected_TAIL
Table S8 PolyA_Tail_Lengths_TAIL-seq.xlsx

data_processing:
Library strategy: TAIL-seq
RNA-seq data was aligned to the human hg19 genome build using Olego and Star aligners, and alternative splicing and alternative polyadenylation were estimated as described below.
Quantas software as described in Charizanis et al, 2012, Neuron, was used to estimate alternative splicing. Olego aligned alignment files were used to count observed junction reads for each exon. Weighted number of exon or exon-junction fragments uniquely supporting the inclusion or skipping isoform of each cassette exon and a probability score was assigned to each isoform. A Fisher’s exact test was used to evaluate the statistical significance of splicing changes using both exon and exon-junction fragments, followed by Benjamini multiple testing correction to estimate the false discovery rate (FDR). In addition, inclusion or exclusion junction reads were used to calculate the proportional change of exon inclusion (dI). See documentation at http://zhanglab.c2b2.columbia.edu/index.php/Quantas_Documentation.
Tandem UTR isoform analysis was performed with the MISO algorithm v0.5.2 using default settings as described in Katz et al, Nature, 2010, except for use of custom 3′UTR isoform annotations. We used a Bayes-factor threshold of 10,000 and difference values (delta Psi) with an absolute value of at least 0.03
For TAIL-seq data analysis, image files were downloaded from the MiSeq and run on tailseeker (Chang et al, 2014, Mol Cell) to determine accurate polyA tail lengths, yielding paired fastq files corresponding to the 5' (R5) and 3' (R3 polyA tail) ends of each read. Reads were aligned against the human genome (hg19) and viral genomes (Human_Herpesvirus_5_strain_Merlin, AY446894.2) using STAR under default parameters. Features were assigned using Subread with gencode v19 annotations and with Human_Herpesvirus_5_strain_Merlin features, and filtered to obtain only the uniquely mapped protein coding genes. For analysis of virally mapped reads, all genes were counted. Reads with tails measuring 0 lengths were removed. For genes with at least 20 mapped reads, median lengths were measured and the global distributions of these lengths were compared against each other using the Kolmogorov-Smirnov test. For each gene captured in all samples and with at least 20 mapped reads, individual tail length distributions were compared amongst samples using the Mann Whitney U test with a P value cutoff of 0.025
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph
Supplementary_files_format_and_content: TableS3: Significantly changing alternative host polyadenylation events
Supplementary_files_format_and_content: TableS6: Significant host gene alternative cassette splicing changes upon CPEB1 KD and CPEB1 OE from RNA-seq
Supplementary_files_format_and_content: TableS8: PolyA tail lengths analyzed by TAIL-seq

value: HFF cells

tag: infection

value: HCMV infected

value: Non-Targeting siRNA treatment


title: Day 16 conceptus_D16-2

description: Sample name: D16-2


title: Day 14 conceptus_D14-1

description: Sample name: D14-1


title: Mock infected Non-targeting siRNA treated human foreskin fibroblasts [Tail-Seq]

description:
Uninfected_TAIL
Table S8 PolyA_Tail_Lengths_TAIL-seq.xlsx

tag: cell type

value: Mock Infected (serum with no HCMV)


title: Hoxa1 ESC rep1

status: Public on Sep 16 2016

city: Utrecht

first: Jacqueline

last: Deschamps

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

data_processing:
The initial step in the 4C-seq analysis is the alignment of the sequencing reads to a reduced genome of sequences that flank HindIII sites (4C fragment ends), using custom Perl scripts. Due to their ambiguous nature in reporting contacts, repetitive fragment ends were excluded from subsequent analysis. The reduced genome was based on mouse genome mm9.
Wiggle tracks contain uniquely mapped reads at 4C fragment ends on the cis chromosome, i.e. the chromosome on which the bait/viewpoint fragment is located.  Scores represent the number of reads mapping to a HindIII restriction fragment end.
Genome_build: mm9

source_name: ESC

growth_protocol: Standard ESC culture in feeder-free conditions, on gelatin-coated dishes, in standard ES medium conditioned on Buffalo Rat Liver (BRL) cells (60%), supplemented with LIF and β-mercaptoethanol)

extract_protocol:
The initial steps of the 4C procedure, as published before (Simonis et al., 2006, Nature Genetics (PMID 17033623)), remain unchanged. Cells are cross-linked using 2% formaldehyde for 10 min at room temperature in PBS 10% FCS and nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated. After reversal of the cross-links, the DNA is purified and treated with the second restriction enzyme treatment (Csp6I). After a second re-ligation step, the sample is purified and a 4C PCR is performed.
4C PCR

value: ESC

tag: experiment type

tag: wnt stimulation


title: LID11

value: Retina, RPE, choroid

value: male

value: Post-hatch day 6

value: 1 day (post-lensing)

tag: lens type


title: GDDH13_fruit2_9DAP

city: Beaucouzé

first: Jean-Marc

last: Celton

address: 42 rue Georges Morel

country: France

institute: INRA-IRHS

data_processing:
methylation call performed with BSMAP v 2.89
Genome_build: MJAX00000000
Supplementary_files_format_and_content: text files with methylation ratios.

organism: Malus domestica

source_name: fruits

growth_protocol: The GDDH13 tree was grown in the orchard following common cultural practices

extract_protocol:
Following liquid nitrogen grinding, DNA was purified using the Machery-Nagel NucleoSpin plant II DNA extraction kit
Bisulfite treatment was applied to determine the cytosine methylation status using the Epitect bisulfite kit. Illumina library was then constructed following manufacturer's instructions

value: fruits collected 9 days after pollination

tag: cultivar

value: 'Golden Delicious' doubled haploid #13 (GDDH13)


title: Day 12 conceptus_D12-5

description: Sample name: D12-5

value: conceptus

tag: day of pregnancy

tag: isolation method


title: LID103

value: Leghorn/New Hampshire

value: Post-hatch day 8

value: 3 days (post-lensing)

value: No Lens


title: LID104


title: LID102


title: GDDH13_leaf

source_name: leaves

value: young leaves


title: Day 12 conceptus_D12-3

description: Sample name: D12-3

value: whole tissue

tag: rna processing

value: Ribosome depleted RNA


title: GDDH13_fruit1_9DAP


title: HCMV infected siCPEB1 siRNA treated human foreskin fibroblasts [Tail-Seq]

description:
HCMV_infected_CPEB1-siRNA treated_TAIL
Table S8 PolyA_Tail_Lengths_TAIL-seq.xlsx

value: HFF cells

value: HCMV infected

tag: treatment

value: CPEB1 siRNA treatment


title: Day 12 conceptus_D12-1

description: Sample name: D12-1


title: LID100

value: Retina, RPE, choroid


title: LID1

value: male

value: Positive (10 diopter)


title: Ades6 wt 0h

source_name: EpiSCs

growth_protocol: Pre-streak embryos (E6.0) were isolated in M2 medium; extraembryonic tissue and surrounding primitive endoderm was removed. Explants were put on MEFs and cultured in presence of Wnt inhibitor IWP2 (2 µM, Sigma Aldrich, dissolved in DMSO) in medium containing 20% KOSR, 77% DMEM/F12, 1x penicillin/streptomycin (100 units/mL, Thermo Fisher Scientific), 2 mM L-glutamine, 1x non-essential amino acids, 0.01% β-mercaptoethanol, Fgf-basic (12 ng/mL, ThermoFisher Scientific ), Activin A (20 ng/mL, R&D Systems).  MEF-free culture took place on surface coated by fibronectin (ThermoFisher Scientific) in medium containing 48% DMEM/F12, 48% Neurobasal Medium 1% B27 Supplement, 0.5% N2 Supplement B (all Thermo Fisher Scientific), 1x penicillin/streptomycin, Fgf-basic (12 ng/mL), Activin A (20 ng/mL), IWP2 (2 µM), 0.01% β-mercaptoethanol. Medium was refreshed on daily basis.

treatment_protocol: Wnt stimulation was done after at least two passages feeder-free culturing by withdrawal of IWP2 and addition of Wnt3a (Cell Guidance Systems, 125 ng/mL) or Chiron (3 µM).

value: EpiSCs

tag: antibody


title: Ades5 wt 0h


title: Hoxa1 ESC rep2

value: ESC


title: Hoxa1 wt 0h rep2

value: epiblast stem cells (EpiSCs)


title: Hoxa1 wt 0h rep1


title: Hoxa13 wt 72hChiron


title: Ades5 wt 48hChiron


title: Ades34 wt 48hChiron


title: Hoxa10 wt 72hChiron


title: Ades5 wt 24hChiron


title: Ades34 wt 24hChiron


title: Hoxa13 wt 48hChiron


title: tagmentation_hek293DMSfxr2_DrosophilaUToskar_gurken

first: Silvi

last: Rouskin

address: 455 Main Street

institute: Whitehead Institute

data_processing:
Illumina bcl2fastq v2.17 was used for base-calling.
Alignments were performed using Tophat v2.1.0 with bowtie2 and the following options for 50bp sequencing run: --no-novel-juncs -N 5 --read-gap-length 7 --read-edit-dist 7 --max-insertion-length 5 --max-deletion-length 5 -g 3
Non-uniquely mapping reads were removed. Additionally, 2nt or 5nt were removed from the 5' end of each read for TGIRT and SSii data, respectively, due non-template addition during reverse transcription.
Mismatch locations were called, and mismatches located within 3nt of an insertions or deletion were masked for future analysis.
In the case of data with a unique molecular index (UMI), sequencing reads were filtered for length >100nt and for unique reads only. For data generated with NexteraXT tagmentation, alignments to both strands were combined.
Genome_build: Saccharomyces cerevisiae assembly R64 (UCSC: sacCer3) or, for human data, the longest RefSeq isoforms from hg19.
Supplementary_files_format_and_content: Tab-delimited files have three columns, representing the name of the aligned sequenced (either chromosome or transcript name), the position within that sequence, and the ratiometric DMS-MaPseq value calculated as the number of mismatches divided by the total sequencing depth at each postion. The position number directly indicates the DMS modified position starting at 1.

organism:
Drosophila melanogaster
Homo sapiens

source_name: Drosophila oocytes, Hek293 cells

growth_protocol: For the in vivo DMS treatment of S. cerevisiae, DMS was added directly to the growing log phase yeast culture at 5% for 3-4 min. 25% BME, 30% isoamyl alcholol was used to quench the DMS. Cells were lysed with SDS, and RNA was isolated with hot acid phenol. For denatured samples, RNA was first extracted and then heated to 95deg for treatment with DMS.

extract_protocol:
Total RNA was extracted using Trizol, and treated with DNase. rRNA was depleted using RNaseH, and tRNA species were depleted using size exclusion columns. A gene-specific primer was used for reverse transcription with the TGIRT enzyme. Gene-specific PCR primers were used to amplify the region of interest, and then dsDNA was fragmented and prepared for sequencing using NexteraXT (Illumina).
Gene specific RT/PCR followed by Nextera XT or LMPCR.
For genome-wide libraries we used 3' ligation, followed by circularixation, and PCR to add the sequencing adapters.

treatment_protocol: Cells  or oocytes were treated with DMS for 5mins, total RNA was extracted with Triazol, rRNA was subtracted with Ribozero (yeast and mammalian cells) or RNaseH with specific oligoes (Drosophila)

tag: strain

value: OreR

tag: cell line

value: HEK 293T


title: Hoxa6 wt 48hChiron

tag: tissue


title: Hoxa6 wt 72hChiron


title: Ades2 wt 12hChiron


title: Ades34 wt 0h


title: Hoxa13 wt 0h


title: Hoxa3 wt 24hChiron


title: Ades6 wt 24hChiron


title: Ades2 wt 0h


title: Hoxa10 wt 48hChiron


title: Hoxa1 wt 48hChiron


title: Hoxa10 wt 0h


title: Hoxa10 wt 24hChiron


title: Hoxa6 wt 24hChiron


title: Hoxa3 wt 48hChiron


title: Hoxa3 wt 0h


title: ATAC-seq wt E6.0 rep1

data_processing:
Adapter trimming: Cutadapt (1.8.1), parameters: -a CTGTCTCTTATA -A CTGTCTCTTATA
Mapping: Bowtie2 (2.1.0), parameters: -X 2000 -p 8
Read cleanup: Samtools (0.1.19), SAM files were converted to BAM files with and duplicate reads were removed with rmdup
Read cleanup: Samtools (0.1.19) reads that were not mapped, paired, uniquely mapped (MAPQ > 1) or mapped to chrM, were removed
Peak calling: MACS2 (2.1.0), parameters: callpeak -B -p 1e-5 -g mm –SPMR –nomodel –llocal 100000
Genome_build: mm9

source_name: E6.0 mouse embryo

extract_protocol:
According to Buenrostro et al. (2013)
Modified version of the Illumina Nextera DNA Sample prep kit.

value: E6.0 mouse embryo

value: ATAC-seq


title: Hoxa1 wt 0h


title: ATAC-seq wt E7.5posterior

source_name: E7.5 posterior part mouse embryo

value: E7.5 posterior part mouse embryo


title: Hoxa1 wt 72hChiron


title: Ades2 wt 48hChiron


title: HepAD38_1

city: Yokohama

first: Kosuke

last: Hashimoto

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

department: IMS

country: Japan

institute: RIKEN

description: 3 days in production medium

data_processing:
Basecalls performed using CASAVA version 1.8
FASTQ files were splitted by barcode using FASTX-Toolkit
Ribosomal RNAs were identified and removed using rRNAdust.
Reads were aligned to the human or mouse genomes using BWA v0.7.5a
Unmapped reads were aligned to HBV genome using BWA 0.7.5a
Transcription start sites (CAGE peaks) on HBV genome were identified using Paraclu.
Genome_build: hg19/GRCh37 for human, mm9/NCBI37 for mouse, and GQ358158.1 for HBV
Supplementary_files_format_and_content: Tab-delimited text file in the bed-like format including normalized expression values (tpm) of HBV promoters for 11 samples. This series contains CAGE peaks only for the HBV genome.

source_name: hepatoma cell line derived from HepG2

extract_protocol:
Total RNA was prepared using TRIzol reagent (Invitrogen) and TURBO DNase (Ambion).
Total RNA was used to synthesize cDNA with random primers. Full-length cDNAs were biotinylated and captured by streptavidin-coated magnetic beads. The cDNAs were released from cap-trapped RNAs, ligated to 5' linkers including barcode sequences. The detailed protocol is described in Ref [PMID:24927836]

value: cell line


title: HepAD38_2

description: 3 days in production medium


title: ATAC-seq wt EpiSCs

treatment_protocol: Wnt stimulation was done after at least two passages feeder-free culturing by withdrawal of IWP2 and addition of Chiron (3 µM).


title: ATAC-seq wt E7.2 rep2

source_name: E7.2 mouse embryo

value: E7.2 mouse embryo


title: PHH1

description: 7 days after HBV infection

source_name: HBV infected primary human hepatocytes

value: Primary hepatocyte


title: HepG2.2.15_1

description: HBV-engineered HepG2

source_name: HBV-engineered HepG2


title: Loucy_WCE_ChipSeq

source_name: T-ALL

growth_protocol: Cells were maintained under typical conditions in RPMI +10% FBS.

value: Loucy

tag: tissue source

value: Blood

tag: chip antibody

value: none (input)


title: ATAC-seq wt E7.2 rep1


title: ATAC-seq wt EpiSCs Chiron48h


title: Hoxa6 wt 0h

value: IWP2 (2 µM)


title: PHH3

description: 7 days after HBV infection


title: Hoxa1 wt 24hChiron


title: MOLT4_input_15309

growth_protocol: Maintained as per ATCC guidelines.    RPMI Glutamax, 10% FBS, PS


title: mRNA-seq_48hr_CX-5461_rep2_haib3866_1

city: Boston

first: Jessica

middle: L

last: Woolnough

address: 77 Avenue Louis Pasteur

institute: Brigham and Women's Hospital

data_processing:
RNA-seq datsets were aligned to hg19 using Tophat2 using default conditions.
Differential gene expression for mRNA-seq datasets was determined by running DEseq2 in R Statistical Environment.
Data file which was used for DESeq2 analysis is included as processed data file "S1SupportingInfo".
Sample named 'mRNA-seq_48hr_ActivinA_rep2' in count table tab of "S1SupportingInfo" represents a concatenation of raw files: mRNA-seq_48hr_ActivinA_rep2_heflin, mRNA-seq_48hr_ActivinA_rep2_haib3866_1, and mRNA-seq_48hr_ActivinA_rep2_haib3866_2
Sample named 'mRNA-seq_48hr_CX-5461_rep2' in count table tab of "S1SupportingInfo" represents a concatenation of raw files: mRNA-seq_48hr_CX5461_rep2_heflin, mRNA-seq_48hr_CX-5461_rep2_haib3866_1, and mRNA-seq_48hr_CX-5461_rep2_haib3866_2
Genome_build: hg19

source_name: H9 hESC treated with a 50 ng/mL final concentration of CX-5461 for 48 hours

growth_protocol: For CX-5461 treatment ,cells were grown to about 75 – 80% confluency. The cells were washed with PBS and 10 ml of RPMI 1640 (GE Healthcare # SH30027.01) was added with 1X B-27 supplement (life technologies # 17504-044) along with CX-5461 (selleckchem # 52684) to a final concentration of 50 ng/ml for 48 hours.  Old media was removed and replaced with fresh media/CX-5461 at 24 hours.

extract_protocol:
RNA-seq: Trizol followed by 2 rounds of poly A selection.
Libraries were prepared by Illumina's Tru-seq Library Preparation Kit v2.  Paired-end sequencing was carried out on Hi-Seq_v4 (PE50 runs).

tag: passage

value: 50 ng/mL CX-5461 for 48 hours


title: mRNA-seq_48hr_ActivinA_rep2_haib3866_1

source_name: H9 hESC treated with a 50 ng/mL final concentration of Activin A for 6 hours

growth_protocol: For Activin A treatment cells were grown to about 75 – 80% confluency. The cells were washed with PBS and 10 ml of RPMI 1640 (GE Healthcare # SH30027.01) was added with 1X B-27 supplement (life technologies # 17504-044) along with Activin A (sigma # SRP3003) to a final concentration of 50 ng/ml for 48 hours.  Old media was removed and replaced with fresh media/Activin A at 24 hours.

value: 50 ng/mL Activin A for 48 hours


title: HepG2.2.15_2

description: HBV-engineered HepG2


title: PHH2

description: 7 days after HBV infection


title: mRNA-seq_48hr_CX-5461_rep2_haib3866_2


title: HepG2.2.15_3

description: HBV-engineered HepG2


title: mRNA-seq_48hr_CX-5461_rep2_heflin


title: mRNA-seq_6hr_ActivinA_rep3

growth_protocol: For Activin A treatment cells were grown to about 75 – 80% confluency. The cells were washed with PBS and 10 ml of RPMI 1640 (GE Healthcare # SH30027.01) was added with 1X B-27 supplement (life technologies # 17504-044) along with Activin A (sigma # SRP3003) to a final concentration of 50 ng/ml for 6 hours.

value: 50 ng/mL Activin A for 6 hours


title: ChIP-seq_Input_hESC

description:
ChIP'd DNA
hg18plusrDNA custom

data_processing:
Illumina CASAVA version 1.8.2 software was used to create the fastq files from the bcl files for ChIP-seq.  bcl2fastq version 1.8.4 was used for RNA-seq.  ChIP fastq files were filtered using the fastx filter tool to have 80% of the reads with a Qual score of >= 20.  The remaining reads were collapsed using the collapser tool to eliminate all PCR duplicates.  The resulting FASTA file was aligned to a custom hg18 annotation that includes the rDNA consensus repeat as an added chromosome called 'chr24_rDNA' using Bowtie2 under default conditions.  The aligned SAM files were converted to BAM using samtools then to BED format using BEDTools.
RNA-seq datsets were aligned to hg19 using Tophat2 using default conditions.
Differential gene expression for mRNA-seq datasets was determined by running DEseq2 in R Statistical Environment.  Processed tag counts across genes tested are in processed data file 'mRNA-seq_GeneCounts_AllSamples.xlsx'.
Genome_build: mRNA-seq = hg19, ChIP-seq = hg18plusrDNA custom build
Supplementary_files_format_and_content: bed + counts

source_name: H9 hESC

growth_protocol: H9 ESCs were obtained from WiCell as per agreement 15-W0341.  H9 ESCs were cultured on matrigel coated plates (corning #354277) in mTeSR 1 media (stemcell technologies # 05857), washed with DMEM/F12 (GE healthcare # SH3002302) and passaged with accutase (MP Biomedicals # 091000449) and ROCK Inhibitor Y-27632 (BD # 562822).

extract_protocol:
Formaldehyde cross-linked nuclei were sonicated. 5% of sonicated chromatin was was reverse-cross-linked, phenol:chloroform extracted and ethanol precipitated.
Library was constructed using Illuina Tru-seq branched adapters and subjected to 25 PCR cycles


title: mRNA-seq_48hr_ActivinA_rep2_haib3866_2


title: CM-pre-NEI-br2

city: Wuhan

first: Jianmei

last: Long

state: Hubei

address: shizishan street NO.1

country: China

institute: Huazhong Agricultural University

description:
replicate 2 for ovules before nucellar embryo initiation stage
Low Molecular Weight RNA

data_processing:
To obtain clean reads, low quality reads, filtering the contaminants and the adaptor sequences were filtered first.
The clean sRNAs  mapped to Rfam10.1 (ftp://ftp.ebi.ac.uk/pub/databases/Rfam) with no more than two mismatches,which were identified as non-coding RNAs (ncRNA), were excluded.
The leftover high-quality clean reads of GF and PU were mapped to the genome of Citrus Sinensis, while PK and CM were mapped to Clementine genome.
Genome_build: Reference genome sequences of Citrus Sinensis were downloaded from http://citrus.hzau.edu.cn/orange/download/data.php, while Clementine genome sequences were downloaded from http://phytozome.jgi.doe.gov/pz/portal.html.
Supplementary_files_format_and_content: tab-delimited text files include raw count and length for each read

organism: Citrus x clementina

source_name: Ovules_Nour clementina (Citrus Clementine)

extract_protocol:
Total RNA was extracted by Trizol method
Small RNA libraries were constructed for sequencing using standard Illumina protocols

value: Nour

value: ovules

tag: age

value: 3 DAF (days after flowering)


title: mRNA-seq_48hr_ActivinA_rep2_heflin


title: ChIP-seq_UBTF_hESC

description:
ChIP'd DNA
hg18plusrDNA custom

extract_protocol:
Formaldehyde cross-linked nuclei were sonicated and UBTF was purified using indicated antibody and protein A beads.  Immunopurified DNA was reverse-cross-linked, phenol:chloroform extracted and ethanol precipitated.
Library was constructed using Illuina Tru-seq branched adapters and subjected to 25 PCR cycles

value: anti-UBTF (Millipore # MABE195)


title: CM-NEI-br1

description:
replicate 1 for ovules at nucellar embryo initiation stage
Low Molecular Weight RNA

value: 7 DAF (days after flowering)


title: CM-pre-NEI-br1

description:
replicate 1 for ovules before nucellar embryo initiation stage
Low Molecular Weight RNA


title: PK-pre-NEI-br1

organism: Citrus reticulata

source_name: Ovules_Ponkan ‘Huagan No.2’ (Citrus reticulata)

value: Ponkan 'Huagan No.2'


title: CM-NEI-br2

description:
replicate 2 for ovules at nucellar embryo initiation stage
Low Molecular Weight RNA


title: ATAC-seq wt E6.0 rep2

value: E6.0 mouse embryo

value: ATAC-seq


title: mRNA-seq_6hr_CX-5461_3

source_name: H9 hESC treated with a 50 ng/mL final concentration of CX-5461 for 6 hours

growth_protocol: For CX-5461 treatment ,cells were grown to about 75 – 80% confluency. The cells were washed with PBS and 10 ml of RPMI 1640 (GE Healthcare # SH30027.01) was added with 1X B-27 supplement (life technologies # 17504-044) along with CX-5461 (selleckchem # 52684) to a final concentration of 50 ng/ml for 6 hours.

value: 50 ng/mL CX-5461 for 6 hours


title: mRNA-seq_6hr_CX-5461_2


title: mRNA-seq_6hr_ActivinA_rep2


title: mRNA-seq_48hr_CX-5461

description:
mRNA
hg19


title: mRNA-seq_48hr_ActivinA

description:
mRNA
hg19


title: mRNA-seq_hESC_rep3


title: ActD_2h_TSA

city: Heidelberg

first: Georg

last: Stoecklin

address: Im Neuenheimer Feld 280

institute: German Cancer Research Center

description: RNA purified from HeLa cells after 16 h TSA treatment followed by 2 h Actinomycin D treatment

data_processing:
Basecalling with CASAVA v1.8.2
Reads were mapped with Bowtie to the human Gencode 19 transcriptome as downloaded from the UCSC Genome Browser wgEncodeGencodeBasicV19 table on May 21, 2014, allowing a maximum of two mismatches and reporting all alignments in the best stratum . The sequences of the rabbit b-globin (HBB2) and Firefly luciferase in vitro transcripts were included into the index.
With an in-house developed Perl script, read counts were summarized at the gene level discarding all reads that map to transcript isoforms of more than one gene (as defined by a common gene symbol).
In order to calculate half-lives with R, read counts were normalized to the sum of 18S and 28S rRNA. Linear regression was performed on log-transformed read counts. Half-lives were calculated from the slope of the regression line with the equation: ln2 / -slope. Only genes with a positive half-live and a coefficient of determination (R2) of more than 0.5 in both conditions were used for subsequent analyses.
To test for significant differences between the slopes of the regression line with and without TSA treatment, we performed a t-test.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Excel sheet that contains for each gene read counts of all samples, half-life with and without TSA treatment, coefficient of determination of the linear regression and a p-value for differences in slope between TSA treatment and control.

growth_protocol: HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (PAA Laboratories), 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in 5% CO2.

extract_protocol:
RNA was purified with the EURx GeneMATRIX universal RNA purification kit.
RNA libraries were prepared for sequencing using the NEBNext Ultra Directional RNA Library Prep Kit.

treatment_protocol: HeLa cells were treated with TSA (500 nM) for 16 h or left untreated, followed by treatment with Actinomycin D (5 μg/ml) for 0 h, 2 h, 4 h and 6 h.

value: HeLa


title: ActD_4h_ctrl

description: RNA purified from HeLa cells after 4 h Actinomycin D treatment


title: PK-NEI-br2


title: mRNA-seq_hESC_rep2


title: mRNA-seq_6hr_CX-5461

description:
mRNA
hg19


title: ChIP-seq_IgG_hESC

description:
ChIP'd DNA
hg18plusrDNA custom

extract_protocol:
Formaldehyde cross-linked nuclei were sonicated and IgG was purified using indicated antibody and protein A beads.  Immunopurified DNA was reverse-cross-linked, phenol:chloroform extracted and ethanol precipitated.
Library was constructed using Illuina Tru-seq branched adapters and subjected to 25 PCR cycles

value: Mouse IgG (Millipore #12-371)


title: mRNA-seq_hESC

description:
mRNA
hg19


title: PK-pre-NEI-br2


title: Drosophila_oskar_DMS_tagmentation_replicate

source_name: Drosophila oocytes

value: OreR

tag: tissue/cell type

value: oocytes


title: Day 21, Animal 86

city: College Station

first: Leslie

middle: Garry

last: Adams

state: TX

address: 402 Raymond Stotzer Blvd.

department: Veterinary Pathobiology

institute: Texas A&M University

description:
demultiplex
Sample name: 86-21_5_exp_day21

data_processing:
Illumina Casava1.7 software used for basecalling.
splitBC was used to split raw barcoded data to create separate fastq files for each sample as listed in sample section
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using trimmomatic.
Reads  mapped to bovine refGenome UMD3.1 whole genome using bowtie2 v2.2.5  with parameters –end-to-end -D 15 -R 2 -N 0 -L 22 -iS,1,1.15
read alignment counts to transcripts by BedTools Multicov with bovine genomic intervals provided by Illumina's Igenome annotation from NCBI
Count table (count to transcript/gene mapping) created by Seralogix's NextGen Sequencing pipeline to form one matrix file genes in rows and samples in column by sample name declared above.
Differential analysis completed by DESeq2
Genome_build: UMD3.1 bovine
Supplementary_files_format_and_content: MP-12vaccinatedCountTableMatrix.txt is a matrix of unnormalized, unfiltered read counts.  First 3 columns are 1) transcript id, 2) gene symbol, 3) gene description.  The remaining columns are sample data corresponding to sample names from above.
Supplementary_files_format_and_content: MP-12normalizedAbundanceTableMatrix.txt generated by DESeq2  is a matrix of normalized and filtered abundance (read counts).  First 5 columns are 1) Unique Identifier, 2) Probe_ID=transcript ID maps to file MP-12vaccinatedCountTableMatrix.txt, 3) Gene Description; 4) Genbank ID, 5) Gene Symbol.  The remaining columns are the normalized data by sample name defined above in Sample descriptions.

organism: Bos taurus

source_name: MP-12 Vaccinated Cattle

growth_protocol: There was no growth protocol, however whole blood was collected from cattle at DAY 0–7, 10, 14 and 21 and immediately mixed at a 3:5 (blood:buffer) ratio with RNALater (Invitrogen). Samples were stored at –20°C until processing.

extract_protocol:
Whole blood was collected at DAY 0–7, 10, 14 and 21 and immediately mixed at a 3:5 (blood:buffer) ratio with RNALater (Invitrogen). Samples were stored at –20°C until processing. To purify RNA, frozen samples were thawed at 37°C, centrifuged at 2000xG, and supernatant discarded. Remaining cell pellets were subjected to two rounds of treatment with Red Blood Cell Lysing Buffer per manufacturer’s instructions (Sigma). RNA from resulting cell pellet was initially extracted by Trizol (Invitrogen) followed by treatment with RNeasy Kit with on-column DNase digestion (Qiagen). Purified RNA was quantified by spectrophotometry on a NanoDrop (Thermo, USA) and by electrophoresis with a BioAnalyzer 2100 (Agilent, USA). Only samples with an RNA Integrity Number (RIN) greater than 8.0 were used for RNASeq analysis.
All RNASeq sequencing was performed at the Texas A&M Genetics and Bioinformatics Center following manufacturer’s instructions. cDNA was generated from RNA samples of sufficient quantity and quality with Ovation RNA-Seq System (NuGen, USA), sheared by focused ultrasound (Covartis, USA) and made into barcoded (multiplexed) libraries with Encore Rapid Library System (NuGen, USA).  Samples were quantified, diluted, pooled re-analyzed with a Bioanalyzer and loaded onto a Genome Analyzer II High-throughput sequencer (Illumina, USA).

treatment_protocol: All cattle were injected subcutaneously or intramuscularly with the Rift Valley fever MP-12 vaccine strain.

tag: individual

value: Animal 86

tag: cell type

value: circulating lymphocytes

tag: vaccine

value: Rift Valley fever, MP-12 vaccine

tag: vaccine administration

value: intramuscular

tag: time

value: Day 21

tag: serologic response status

value: vaccinated, protected


title: Day 21, Animal 74

description:
demultiplex
Sample name: 74-21_1_exp_day21

value: Animal 74

value: subcutaneous


title: ActD_6h_TSA

description: RNA purified from HeLa cells after 16 h TSA treatment followed by 6 h Actinomycin D treatment


title: ActD_4h_TSA

description: RNA purified from HeLa cells after 16 h TSA treatment followed by 4 h Actinomycin D treatment


title: MRPS21_Ribosnitch

source_name: In vitro transcription

extract_protocol:
dsDNA was in vitro transcribed from each allele, mixed, and folded in vitro by a brief denaturation at 95deg and reannealing at 37deg. 10% DMS was added for 10min, then quenched with BME. The RNA was then ligated using T4 K227Q truncated RNA ligase, and reverse transcribed with the TGIRT enzyme. Circ ligase (Epicentre) was used for circularization, and Illumina adapters were introduced by 8-10 cycles of PCR.
Cells or oocytes were treated with DMS for 5mins, total RNA was extracted with Triazol, rRNA was subtracted with Ribozero (yeast and mammalian cells) or RNaseH with specific oligoes (Drosophila)
Gene specific RT/PCR followed by Nextera XT or LMPCR. For genome-wide libraries we used 3' ligation , followed by circularixation, and PCR to add the sequencing adapters.

treatment_protocol: For the in vivo DMS treatment of S. cerevisiae, DMS was added directly to the growing log phase yeast culture at 5% for 3-4 min. 25% BME, 30% isoamyl alcholol was used to quench the DMS. Cells were lysed with SDS, and RNA was isolated with hot acid phenol. For denatured samples, RNA was first extracted and then heated to 95deg for treatment with DMS.

tag: molecule subtype

value: mRNA


title: Day 14, Animal 86

description:
demultiplex
Sample name: 86-14_5_exp_day14

value: Day 14


title: ActD_0h_TSA

description: RNA purified from HeLa cells after 16 h TSA treatment followed by 0 h Actinomycin D treatment


title: ActD_6h_ctrl

description: RNA purified from HeLa cells after 6 h Actinomycin D treatment


title: PK-NEI-br1

tag: cultivar

value: Ponkan 'Huagan No.2'

value: ovules

value: 7 DAF (days after flowering)


title: Day 10, Animal 88

description:
demultiplex
Sample name: 88-10_4_exp_day10

value: Animal 88

value: Day 10


title: ChIP-seq_UBTF_6hr_ActivinA

description:
ChIP'd DNA
hg18plusrDNA custom

value: anti-UBTF (Millipore # MABE195)

tag: treatment

value: 50 ng/mL Activin A for 6 hours


title: ActD_0h_ctrl

description: RNA purified from HeLa cells after 0 h Actinomycin D treatment

value: HeLa


title: ActD_2h_ctrl

description: RNA purified from HeLa cells after 2 h Actinomycin D treatment


title: Day 21, Animal 76

description:
demultiplex
Sample name: 76-21_2_exp_day21

value: Animal 76

value: circulating lymphocytes


title: Day 7, Animal 86

description:
demultiplex
Sample name: 86-7_4_exp_day7

value: Animal 86

value: Rift Valley fever, MP-12 vaccine

value: intramuscular

value: Day 7


title: HEK 293Ts_DMSvivo_genomeWide

source_name: HEK 293T cells

extract_protocol:
Cells or oocytes were treated with DMS for 5mins, total RNA was extracted with Triazol, rRNA was subtracted with Ribozero (yeast and mammalian cells) or RNaseH with specific oligoes (Drosophila)
Gene specific RT/PCR followed by Nextera XT or LMPCR. For genome-wide libraries we used 3' ligation , followed by circularixation, and PCR to add the sequencing adapters.

value: HEK 293T


title: mRNA-seq_6hr_ActivinA

description:
mRNA
hg19


title: Day 14, Animal 88

description:
demultiplex
Sample name: 88-14_4_exp_day14

value: vaccinated, protected


title: Day 21, Animal 75

description:
demultiplex
Sample name: 75-21_3_exp_day21

value: Animal 75

value: subcutaneous

value: Day 21


title: Day 21, Animal 88

description:
demultiplex
Sample name: 88-21_4_exp_day21


title: Scer_UMI_SFT2

organism: Saccharomyces cerevisiae

source_name: BY4741 cells

extract_protocol:
Total RNA was extracted using hot acid phenol, and treated with DNase. rRNA was depleted using RiboZero (Epicentre), and tRNA species were depleted using size exclusion columns. A gene-specific primer with a 10bp random barcode and common overhang on the 5'end was used for reverse transcription with the TGIRT enzyme. A gene-specific forward PCR primer with a 5'overhang was used with a primer complementary to the RT primer overhang were used to amplify the region of interest. A second round of PCR using the common primer overhangs were using to index samples and add adaptors for sequencing.
Cells or oocytes were treated with DMS for 5mins, total RNA was extracted with Triazol, rRNA was subtracted with Ribozero (yeast and mammalian cells) or RNaseH with specific oligoes (Drosophila)
Gene specific RT/PCR followed by Nextera XT or LMPCR. For genome-wide libraries we used 3' ligation , followed by circularixation, and PCR to add the sequencing adapters.

tag: strain

value: mRNA


title: p19syn_nb.mock.mRNA

city: Budapest

first: <Up loaded by Bioinformatics core  facility>

last: <PI shown in the article>

address: Szigony st 43.

country: Hungary

institute: HUN-REN Institute of Experimental Medicine

description: processed data file: nbv5.datamatrix.txt

data_processing:
adapter removeal (UEA smallRNA workbanch 3.0)
Filtration of low complexety reads and reads under 14 nt (UEA smallRNA workbanch 3.0)
alignmant to genomes (PatMaN 1.2.2)
reads were normalized to 1 million total reads passing quality control
Genome_build: Niben v1.0.1. and NC_003532.1
Supplementary_files_format_and_content: Processed data files were uploaded as data matrixes. These matrixes contain a short header followd by the data itself. Headder lines alway begin with a '#'.  The first column contains the sequences, second column contains total read counts, the third contains normalised read counts. Column are TAB separsted.
Quality control was done by fastQC 0.10.1
Trim_galore 0.4.1 and FASTX Toolkit 0.0.13 were used to remove adaptor sequences, low quality bases, reads under 20 nt and unpaired reads.
Bowtie2 was used to align reads to the transcriptome
reads were normalized to 1 million total reads passing quality control
Genome_build: Nbv5
Supplementary_files_format_and_content: Processed data were uploaded as a data matrix. The matrix contains a short header followed by the data itself. Header lines always begin with a '#'.  The first column contains the transcriptome IDs, second contains wt uninfected read counts from Baksa et al 2015 doi: 10.1186/s12864-015-2209-6, the third contains  read counts from sample p19syn_nb.cym19s.mRNA, the fourth column contains read counts from sample wt_nb.cym19s.mRNA, the fifth contains read counts from sample p19syn_nb.mock.mRNA. Column are TAB separated.

organism: Nicotiana benthamiana

growth_protocol: plants were grown under constant 21 °C, 16h light - 8h dark

extract_protocol:
RNA was extracted with phenol-chlorophorm (as described in the material and methods). RNA was fractionated by isolating from a 8% denaturing poliacrylamide gel run.
RNA-seq: RNA was extracted with phenol-chlorophorm (as described in the material and methods). RNA was polyA fractionated prior library prep.
Illumina TruSeq Small RNA Library Preparation Kit and Illumina TruSeq Stranded mRNA library prep

treatment_protocol: Young leafs of 5-6 week old plants were inoculated. Samples were taken 7 days post inoculation, from upper (non inoculated) leafs.

tag: genotype/variation

value: transgenic p19syn

tag: infection with virus

value: young leaf


title: Day 14, Animal 76

description:
demultiplex
Sample name: 76-14_2_exp_day14

value: Day 14


title: Day 14, Animal 75

description:
demultiplex
Sample name: 75-14_3_exp_day14


title: Day 7, Animal 74

description:
demultiplex
Sample name: 74-7_2_exp_day7

value: Animal 74

value: vaccinated, not protected


title: Day 10, Animal 86

description:
demultiplex
Sample name: 86-10_5_exp_day10

value: Day 10


title: Day 14, Animal 74

description:
demultiplex
Sample name: 74-14_1_exp_day14

value: circulating lymphocytes


title: Day 10, Animal 76

description:
demultiplex
Sample name: 76-10_2_exp_day10

value: Animal 76


title: Day 10, Animal 75

description:
demultiplex
Sample name: 75-10_1_exp_day10

value: Rift Valley fever, MP-12 vaccine


title: PU-NEI-br2

description:
replicate 2 for ovules at nucellar embryo initiation stage
Low Molecular Weight RNA

organism: Citrus maxima

source_name: Ovules_Huanong red pummelo (Citrus grandis)

value: Huanong red

tag: tissue

value: ovules

value: 3 DAF (days after flowering)


title: wt_nb.mock.agoip_ago2ip.biorep.sRNA

value: wild type

tag: immunoprecipitation

value: AGO2 IP

tag: ip antibody

value: custom-made NbAGO2 antibody, generated by immunization of rabbits with the synthetic peptide CLEDPEGKDPPRDVF by GenScript


title: Scer_TGIRT_2andHalf_percent_DMS_vivo_genomeWide_MiSeq

extract_protocol:
Total RNA was extracted using hot acid phenol. mRNA was randomly fragmented using Ambion fragmentation reagents, followed by PNK, and ligation using T4 K227Q truncated RNA ligase. rRNA was depleted post-ligation using RiboZero (Epicentre), and reverse transcription was done with the TGIRT enzyme. Circ ligase (Epicentre) was used for circularization, and Illumina adapters were introduced by 8-10 cycles of PCR.
Gene specific RT/PCR followed by Nextera XT or LMPCR.
For genome-wide libraries we used 3' ligation, followed by circularixation, and PCR to add the sequencing adapters.


title: ChIP: Input_SKNSH_012615 [lab: Look-DFCI]

data_processing:
Aligned using /usr/local/bin/bowtie with configuration -p 4 --best -k 2 -m 2 --sam -l 40
hg19
Supplementary_files_format_and_content: WIG files(s) represent counts of aligned reads within 50 bp bins with each read being extended 200 in the direction of alignment. Counts are in reads-per-million and floored at 0.1

source_name: Neuroblastoma

growth_protocol: According to ATCC guidelines

extract_protocol:
Purified immunoprecipitated DNA were prepared for sequencing according to a modified version of the Solexa Genomic DNA protocol. Fragmented DNA was end repaired and subjected to 18 cycles of LM-PCR using oligos provided by Illumina. Amplified fragments between 150 and 300bp (representing shear fragments between 50 and 200nt in length and ~100bp of primer sequence) were isolated by agarose gel electrophoresis and purified.
Whole cell extracts were sonicated to solubilize the chromatin. The chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using different antibodies.

tag: cell line

value: SK-N-SH

tag: tissue source

value: Brain

tag: chip antibody

value: none (input)


title: Scer_TGIRT_5percent_DMS_vivo_genomeWide_MiSeq


title: GF-NEI-br1

description:
replicate 1 for ovules at nucellar embryo initiation stage
Low Molecular Weight RNA

organism: Citrus x paradisi

source_name: Ovules_Cocktail grapefruit (Citrus paradisi)

value: Cocktail

tag: age


title: Day 6, Animal 86

description:
demultiplex
Sample name: 86-6_5_exp_day6

tag: individual

value: Animal 86

tag: cell type

tag: vaccine

value: intramuscular

value: Day 6


title: Day 6, Animal 76

description:
demultiplex
Sample name: 76-6_3_exp_day6

tag: vaccine administration

value: subcutaneous

tag: time

tag: serologic response status

value: vaccinated, not protected


title: wt_nb.cymrsv.agoip_input.biorep.sRNA

description: input of wt_nb.cymrsv.agoip_ago1ip.biorep and wt_nb.cymrsv.agoip_ago2ip.biorep

value: CymRSV

value: young leaf


title: Scer_UMI_ASH1

tag: molecule subtype

value: mRNA


title: Day 7, Animal 76

description:
demultiplex
Sample name: 76-7_3_exp_day7

value: Animal 76

value: circulating lymphocytes

value: Rift Valley fever, MP-12 vaccine

value: Day 7


title: PU-pre-NEI-br1

description:
replicate 1 for ovules before nucellar embryo initiation stage
Low Molecular Weight RNA

tag: cultivar

value: Huanong red

value: ovules

value: 0 DAF (days after flowering), i.e anthesis


title: p19syn_nb.cym19s.mRNA

tag: genotype/variation

value: transgenic p19syn


title: Input for ChIP:P12-ICHIKAWA_032615 [lab: Look-DFCI]

value: Blood

value: none (input)


title: wt_nb.cym19s.mRNA

value: wild type

tag: infection with virus


title: Day 10, Animal 74

description:
demultiplex
Sample name: 74-10_3_exp_day10

value: Animal 74

value: Day 10


title: GF-NEI-br2

value: Cocktail

tag: tissue

value: 3 DAF (days after flowering)


title: wt_nb.mock.agoip_input.biorep.sRNA

description: input of wt_nb.mock.agoip_ago1ip.biorep and wt_nb.mock.agoip_ago2ip.biorep

value: young leaf

tag: immunoprecipitation


title: PU-NEI-br1


title: ChIP_Input_BE2C_012615 [lab: Look-DFCI]

value: Brain


title: Input for ChIP:PF-382_032615 [lab: Look-DFCI]


title: Day 7, Animal 75

description:
demultiplex
Sample name: 75-7_1_exp_day7

value: Animal 75

value: subcutaneous

value: vaccinated, not protected


title: PU-pre-NEI-br2

description:
replicate 2 for ovules before nucellar embryo initiation stage
Low Molecular Weight RNA


title: Day 6, Animal 88

description:
demultiplex
Sample name: 88-6_4_exp_day6

value: Animal 88

value: circulating lymphocytes

value: Rift Valley fever, MP-12 vaccine

value: intramuscular

value: Day 6


title: gurken_tagmentation

tag: strain

value: OreR

tag: tissue/cell type

value: oocytes


title: GF-pre-NEI-br1

value: ovules

value: 0 DAF (days after flowering), i.e anthesis


title: wt_nb.cym19s.agoip_input.biorep.sRNA

description: input of wt_nb.cym19s.agoip_ago1ip.biorep and wt_nb.cym19s.agoip_ago2ip.biorep

value: wild type

value: none (input)


title: Day 5, Animal 88

description:
demultiplex
Sample name: 88-5_3_exp_day5

value: Day 5


title: wt_nb.cymrsv.agoip_ago2ip.biorep.sRNA

value: CymRSV

value: young leaf

value: AGO2 IP

tag: ip antibody

value: custom-made NbAGO2 antibody, generated by immunization of rabbits with the synthetic peptide CLEDPEGKDPPRDVF by GenScript


title: Day 6, Animal 75

description:
demultiplex
Sample name: 75-6_1_exp_day6

value: Animal 75

value: subcutaneous

value: vaccinated, not protected


title: 293T_positiveControlsTagmentation

tag: cell line

value: HEK 293T

value: mRNA


title: wt_nb.cym19s.agoip_ago1ip.biorep.sRNA

value: AGO1 IP

value: custom-made NbAGO1 antibody raised against the peptides MVRKKRTDVPGGAESC and CHLGKRLPAYDGRKSL in rabbits by Eurogentec


title: Day 5, Animal 76

description:
demultiplex
Sample name: 76-5_1_exp_day5

value: Animal 76

value: circulating lymphocytes

value: Rift Valley fever, MP-12 vaccine


title: wt_nb.mock.agoip_ago1ip.biorep.sRNA

value: wild type


title: Scer_TGIRT_DMS_denatured_genomeWide


title: GF-pre-NEI-br2

value: Cocktail

value: ovules

tag: age

value: 0 DAF (days after flowering), i.e anthesis


title: Day 6, Animal 74

description:
demultiplex
Sample name: 74-6_2_exp_day6

tag: individual

value: Animal 74

value: Day 6


title: GM12878 oxaliplatin XR-seq Rep2

city: Jerusalem

first: Sheera

last: Adar

state: Israel

address: Botnar Building Room 001, Faculty of Medicine, Hebrew University, Ein Kerem

department: Department of Microbiology and Molecular Genetics

country: Israel

institute: Hebrew University of Jerusalem

description: Excised oligomer released in Nucleotide Excision Repair (XR-seq)

data_processing:
Library strategy: XR-seq
Base calling using Casava v1.8.2. Demultiplexing performed allowing 1 mismtach in the index
Damage-seq
Reads from undamaged DNA were identified as starting with the sequence 5’-GACTGGTTCCAATTGAAAGTGCTCTTCCGATCT-3’ and filtered with cutadapt (Martin M. 2011)
Paired reads were aligned to the hg19 human reference genome using bowtie (Langmead et al. 2009) with the command options   –q --nomaqround --phred33-quals –S –X 1000 –m 4 –seed 123.
PCR artifacts were filtered by only retaining unique alignments based on both paired reads.
Damage position and nucleotide composition were then determined as the 2 nt  upstream of the first read start using samtools and bedtools
Wiggle base counts files of 25bp fixed step windows were created for the merged biological replicates from the bed files. The count data was normalized for total sequencing depth on each chromosome. Wiggle files were d transferred to bigWig format for viewing on the UCSC browser.
XR-seq
Flanking adapter sequences were removed from the reads using trimmomatic (Bolger et al. 2014).
Reads were aligned to the hg19 human reference genome using bowtie (Langmead et al. 2009) with the command options  -q --nomaqround --phred33-quals -m 4 -n 2 -e 70 -l 20 --best –S
Aligned readsfor each biological replicate were split into “PLUS” and “MINUS” strands, transformed to bed format, and sorted to remove duplicate reads.
Wiggle base counts files of 25bp fixed step windows were created for each of the replicates from the bed files. The count data was normalized for total sequencing depth on each chromosome. Wiggle files were d transferred to bigWig format for viewing on the UCSC browser.
Genome_build: hg19
Supplementary_files_format_and_content: There are bigWig files of normalized 25bp fixedStep count data for both biological replicates, split into plus and minus strands

source_name: B Lymphocytes

growth_protocol: NHF1 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 2 mM glutamine at 37 °C in a 5% CO2 humidified chamber. GM12878 cells were cultured in RPMI 1640 medium (without phenol red) supplemented with 15% fetal bovine serum at 37 °C in a 5% CO2 humidified chamber.

extract_protocol:
Cell pellets were resuspended in 900 µL cold lysis buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA, 250 mM NaCl, 0.5% TritonX-100 and 0.1% SDS) with 10 µL RNaseA (Sigma) and incubated on ice for at least 10 min. Lysates were sonicated by Misonix Sonicator 3000 with microtip on ice water to generate fragments averagely 400 bp in length and then centrifuged at 14,000 rpm for 10 min to pellet debris. The supernatant was incubated with 10 µL Proteinase K (New England Biolabs) at 55°C for 30 min, followed by phenol/chloroform extraction and ethanol precipitation. DNA pellet was dissolved in 100 µL 1xTE buffer, and fragments 200-700 bp in length were selected by 0.5x/0.7x (50/70 µL) HighPrep PCR beads (MagBio) according to manufactory’s guidelines. DNA concentration was determined by NanoDrop 1000 (Thermo).  For naked DNA samples, DNA fragments were prepared from untreated GM12878 cells as described above. For untreated samples, 1 µg size-selected DNA were used to prepare library by NEBNext DNA preparation kit following the manufactory’s instruction (not Damage-seq process). For UV treatment, 5 µg DNA in 50 µL 0.1xTE were irradiated with 20J/m2 UVC as described and subjected to Damage-seq. For cisplatin treatment, 5 µg DNA were incubated with 20 µM cisplatin in a final volume of 50 µL at room temperature for 15 min. Treated DNA were purified through a G50 spin column (GE) immediately and subjected to Damage-seq.
For damage seq:   5 µg size-selected DNA were used for End-repair and dA-tailing by NEBNext DNA preparation kit following manufactory’s instruction. Ad1 ( oligos 5’-phos-GATCGGAAGAGCACACGTCTGAACTCCAGTCA-SpC3 and 5’-NNNNNGACTGGTTCCAATTGAAAGTGCTCTTCCGATC*T, 500 pmol) was ligated to both ends by NEBNext Quik Ligase for more than 12 hr under 16°C, then purified by HighPrep PCR beads and eluted in 126 µL 0.1x TE. Damaged DNA immunoprecipitation was performed as described previously (Hu et al. Genes and Development 2015) with modification. To denature DNA, 42 µL 8M Urea were added (to a final concentration of 2M), followed by boiling for 2 min and immediately putting on ice water for 2 min. Then 50 µg denatured sonicated salmon sperm DNA (Stratagene) and 25 µL 8x IP Buffer (for (6-4)PP and CPD, see below) or 8x PEXB Buffer (for cisplatin and oxaliplatin, see below) were added, followed by incubation with antibody-coated beads which were prepared as described below. For CPD, samples were washed three times with IP Buffer (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 0.5% sodium deoxycholate), then samples were incubated with 5 µL of protein G Dynabeads (10003D, Thermo) slurry and 5 µL of anti-rabbit Dynabeads (11203D, Thermo) slurry with 10 µg carrier DNA, 1 µL of rabbit anti-mouse IgG ((ab46540, Abcam) and 1 µL of anti-CPD (Kamiya Biomedical) antibody in 25 µL of IP Buffer for 3 hrs at 4°C. For (6-4)PP, 10 µL of each Dynabeads, 20 µg denatured sonicated salmon sperm DNA, 2 µL of rabbit anti-mouse IgG and 2 µL of anti-(6-4)PP (Cosmo Bio) antibody were used for one reaction as described above. For cisplatin and oxaliplatin-treated sample, PEXB Buffer (1xPBS, 2 mM EDTA, 0.01% Triton X-100 and 0.025% BSA) was used instead of IP Buffer. Each reaction was incubated with 40 µL of anti-rat Dynabeads (11035, Thermo) slurry and 1.5 µL anti-cisplatin modified DNA antibody (ab103261, Abcam). After incubation, beads were washed by IP Buffer or PEBX Buffer and incubated with denatured DNA overnight at 4°C.   For (6-4)PP or CPD samples, the beads were then washed sequentially with Wash Buffer I (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 1% Triton X-100, and 2M Urea), Wash Buffer II (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, and 0.1% SDS), Wash Buffer III (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 150 mM LiCl, 1% NP-40, and 1% sodium deoxycholate), Wash Buffer IV (100 mM Tris-Cl pH 8.0, 1 mM EDTA, 500 mM LiCl, 1% NP-40, and 1% sodium deoxycholate) and TE (10 mM Tris-Cl pH 8.0 and 1 mM EDTA) at room temperature. For cisplatin and oxaliplatin samples, the beads were washed sequentially with PEXU Buffer (1xPBS, 2 mM EDTA, 0.01% Triton X-100 and 1.6M Urea), PEX Buffer (1xPBS, 2 mM EDTA, 0.01% Triton X-100), IP Buffer and TE. The fragments containing damage were eluted by incubation with 100 µL of Elution Buffer (50 mM NaHCO3, 1% SDS) at 65°C for 10 min. The eluted DNA was then isolated by phenol/chloroform extraction followed by ethanol precipitation.  NEBNext Q5 Hot Start HiFi PCR Master Mix was used for primer extension in the presence of purified DNA and 30 pmol Bio3 (5’-bio-TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, without subtractive hybridization) or Bio3U (5’-bio-AGAGTG/dU/GACTGGAGTTCAGACGTGTGCTCTTCCGATCT, with subtractive hybridization) in a thermocycler for 45s at 98°C followed by 5 min at 65°C. Then 2 µL of Exo1 (New England Biolabs) were added to degrade excessive primers at 37°C for 10 min. After HighPrep PCR beads purification, DNA was denatured by boiling for 2 min and immediately putting on ice water for 2 min, then incubated with 5 µL of Dynabeads MyOne Streptavidin C1 (Thermo) in 30 µL of 1x B&W Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl) at 4°C for 1 hr. For Bio3 primer, biotinylated DNA were eluted by a short incubation (~10s) in 100 µL of nonionic water at 75°C and concentrated by ethanol precipitation.   For Bio3U primer, DNA were released by incubation with 2 µL of USER Enzyme (New England Biolabs) and 1 µL of FastAP (Thermo) in 15 µL of 1x CutSmart Buffer (New England Biolabs) for 20 min at 37°C. The beads were washed with 15 µL of 1x B&W Buffer and both supernatants were combined. For subsequent subtractive hybridization, 30 pmol of oligo SH (5’-bio-NNGACTGGTTCCAATTGAAAGTGCTCTTCCG-SpC3) were added and followed by boiling for 2 min and naturally cooling down to room temperature. Oligo SH was then captured by incubating with 15 µL of Dynabeads MyOne Streptavidin C1 in 50 µL of 1x B&W Buffer. After separating the liquid on magnet, the beads were washed with 60 µL of 1x B&W Buffer and both supernatants were combined. DNA was then isolated by phenol/chloroform extraction followed by ethanol precipitation.  To add the second adaptor to the 3’ end, purified DNA were incubated with 40 pmol of Ad2 (oligos 5’-phos-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-SpC3 and 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNN-SpC3) in 10 µL of 1x Hybridization Buffer for 10 min at 65°C and then for 5 min at 16°C in a thermal cycler. To perform ligation, 4 µL of 5x ligase buffer, 1 µL of T4 DNA ligase HC (Thermo), 1 µL of 50% PEG8000 (New England Biolabs), and 4 µL of H2O were added to each reaction. The reactions were incubated overnight at 16°C.   After HighPrep PCR beads purification, ligated DNA were PCR-amplified by NEBNext Q5 Hot Start HiFi PCR Master Mix for 11-13 cycles with NEBNext Multiplex Oligos for Illumina (New England Biolabs). The PCR products were purified by HighPrep PCR beads and concentration was determined by Pico Green (Thermo).
XR-seq libraries were prepared as described (Hu et al. 2015) with modifications in the damage-specific immunoprecipitation and in vitro reversal steps. GM12878 cells were treated with 200 µM cisplatin or oxaliplatin for 3 hr and collected as described above. Cells were lysed and primary excision products were pulled down by TFIIH co-immunoprecipitation and followed by adaptor ligation on both ends. The damage-specific immunoprecipitation with anti-cisplatin modified DNA antibody was performed as described in Damage-seq with minor difference. For one reaction, 10 µg of denatured sonicated salmon sperm DNA, 25 µL of anti-rat Dynabeads slurry and 1 µL of anti-cisplatin modified DNA antibody were pre-incubated in 100 µL of 1x PEXB buffer at 4°C for 2-4 hours. Pre-incubated beads were then incubated with ligation products and 20 µg of denatured sonicated salmon sperm DNA in 100 µL of 1x PEXB buffer at 4°C overnight. After sequential wash w

treatment_protocol: UV irradiation of NHF1 cells was performed as described previously(Gaddameedhi et al. 2010). Briefly, culture medium was removed from ~90% confluent cells in one R150 dish, then cells were irradiated under a GE germicidal lamp emitting primarily 254-nm UV light (1 J/m2/s) connected with a digital timer for 20s (20 J/m2 in total). Following irradiation, the dish was put on ice immediately, and the cells were washed once by ice-cold PBS and harvested with a cell scraper in PBS and collected by centrifugation. GM12878 cells were grown to ~106 cells/ml in colorless medium. 10ml medium (~107 cells) were transferred to one R150 dish and irradiated as described above. Then cells were immediately transferred to a pre-chilled 15 ml tube on ice, collected by centrifugation, washed by ice-cold PBS. Cell pellets were frozen at -80°C.   To treat the cells with cisplatin or oxaliplatin, fresh stocks were made every time before treatment. The drug was dissolved in DMSO to 20 mM and immediately added to medium to a final concentration of 200 µM. Cells were further incubated at 37°C for 1.5 hour. Then both cells were harvested as described above.

tag: source

value: Coriell Institute

tag: uv exposure

tag: time after irradiation


title: wt_nb.cymrsv.agoip_ago1ip.biorep.sRNA

value: CymRSV

value: young leaf


title: wt_nb.mock.direct

description:
Nicotiana benthamiana
smallRNA-seq

data_processing:
adapter removeal (UEA smallRNA workbanch 3.0)
out filtration of low complexety reads and reads under 14 nt (UEA smallRNA workbanch 3.0)
alignmant to genomes (PatMaN 1.2.2)
normalization, 5' end analysis and visualization programs have been uploaded to: https://github.com/kontral/Burgyan_Lab to ensure reproducibility and transparency.
Genome_build: Niben v1.0.1 and ViralProj15066 (NC_003532.1)
Supplementary_files_format_and_content: Processed data files were uploaded as data matrixes. These matrixes contain a short header followed by the data itself. Header lines alway begin with a '#'.  The first column contains the sequences, second column contains total read counts, the third contains normalized read counts. Columns are TAB delimited.

extract_protocol:
Immunoprecipitation RNA was extracted with phenol-chloroform (as described in the material and methods). RNA was fractioned purified by isolating from a 8% denaturing poliacrylamide gel.
Standard Illumina TruSeq Small RNA Library Preparation Kit

treatment_protocol: Young leafs of 5-6 week old plants were inoculated as described. Samples were taken 7 days post inoculation, from upper leafs.

tag: plant genotype

value: mock


title: Input for ChIP:CCRF-CEM_032615 [lab: Look-DFCI]

first: Richard

last: Young

address: 9 Cambridge Center

institute: Whitehead Institute for Biomedical Research

value: CCRF-CEM

tag: tissue source

value: Blood

tag: chip antibody

value: none (input)


title: Input for ChIP:DU.528_032615 [lab: Look-DFCI]