title: MAC_heavy_MNase_rep2

first: Yifan

last: lIU

address: 1301 Cathrine street

description: Heavy MNase treatment replicate 2

data_processing:
MNase-seq reads of each sample were firstly mapped to the genome assembly of T. thermophila using TopHat2 allowing 2 edit distances (including mismatch and indel). For MAC MNase-seq data, the reads were mapped to the newest MAC genome assembly in Tetrahyemna genome database (TGD) (http://ciliate.org/index.php/home/downloads) ; for MIC MNase-Seq data, reads were mapped to the MIC genome assembly of T. thermophila (http://www.broadinstitute.org/annotation/genome/Tetrahymena/MultiHome.html).
Unique mapped reads were extracted, and potential PCR duplicates (read pairs with same location in genome) were removed and kept only one. To ensure the reads are generated from mono-nucleosome, only fragments (a fragment, corresponded to the insert DNA fragment in library preparing, defined by a pair of properly mapped reads) length form 120-260bp were selected to subsequent analysis.
The mapped reads were visualized using Gbrowse2.Read counts were calculated using featureCounts software.
Genome_build: Tetrahyemna thermophila MAC genome assembly and MIC genome assembly (http://ciliate.org/index.php/home/downloads)
Supplementary_files_format_and_content: Called peaks with bed format
Supplementary_files_format_and_content: Reads counts in dyad±20bp and dyad±73bp of well positioned nucleosomes
Supplementary_files_format_and_content: Mapped 120-260bp MNase fragments (read pairs) with bed format,three columns are Scaffold_ID, Start_position, End_position.

organism: Tetrahymena thermophila CU428

source_name: macronucleus (MAC)

growth_protocol: 1.6 L of Tetrahymena thermophila wild-type strain CU428 was grown in 1xSPP at 30 degree to a density of ~ 200K cells/ml.

extract_protocol:
Mononucleosomal DNA was purified using two methods: agarose gel electrophoresis and sucrose gradient.     For  gel electrophoresis, Add 10 mM EGTA, 1 mM EDTA and 1 % [v/v] SDS and sit at RT for 5 min to stop reaction. DNA was purified through phenol-chloroform extraction after RNAse A and ProK treatment, then ethanol-precipitated, and resuspended in ddH2O. 2 ul of DNA from each sample was run on a 2 % agarose-TAE gel to check the progression of MNase digestion. Mononucleosomal DNA was subsequently purified by Gel DNA recovery kit (Zymoclean) after collecting the proper agarose gel band;     For  sucrose gradient, Add 10 mM EGTA, 1 mM EDTA and 5 mM Na Butyrate to stop reaction. Add 0.1 % [v/v] TX-100 to do nucleus permeabilization and sit on ice for 10 min. Digested DNA with histone was cleaned by centrifugation at 3000 g, 4 oC for 10 min. Mononucleosome was collected by centrifugation at 103000 g, 4 oC for 17 hours in sucrose gradient buffer (10 mM Tris-HCl, 0.1 mM EDTA, 0.1 M NaCl, 5 mM Na Butyrate, with 5%, 10%, 15%, 20%, 25% sucrose, in total 5 sucrose gradient layer, 1.2ml for each layer). Use loading tips (200 ul) to take supernatant, from top to bottom, 200 ul each time to wells of UV-compatible 96-well plate carefully. Measure OD260 & OD280 of each well. According to the light absorption curve, proper fractions containing mononucleosome were collected. Coomassie brilliant blue staining was performed to validate the presence of histones. DNA was subsequently purified through phenol-chloroform extraction after RNAse A and ProK treatment, then ethanol-precipitated, and resuspended in ddH2O. 2 ul of DNA from each sample was run on a 2 % agarose-TAE gel to check the progression of MNase digestion.
Purified nucleosome–sized DNA after MNase digestion was used to prepare Illumina sequencing libraries according to manufacturer’s instructions without the fragmentation step. Paired-end sequencing was peformed using Illumina platform.

treatment_protocol: Cells were collected and resuspended in 200 ml ice-cold medium A (10 mM Tris pH 7.5, 5 mM MgCl2, 0.1 M sucrose, 10 mM butyric acid, 4 % [v/v] gum Arabic, pH to 6.75). MAC were released by blending and collected by differential centrifugation (1500-2000g). Approximately 0.5x108 MAC were digested by MNase (50 mM Tris-HCl (pH 8), 5 mM CaCl2, 1mM β-mercaptoethanol, 0.1% NP-40, 0.1mg/ml BSA, 20-100 unit MNase) at 25 oC for 15 min. MNase digestion was stopped by adding 10 mM EGTA and 1 mM EDTA. Mono-nucleosome DNA was sized selected by agarose gel purification; alternatively, mono-nucleosomes were purified by sucrose gradient ultracentrifugation.

tag: purification method

value: gel electrophoresis


title: MDA_MB_231_DAC

status: Public on Sep 14 2016

type: SRA

city: Lyon

first: Robert

last: Dante

address: 28, rue Laennec

department: Department of Tumoral Escape

country: France

institute: Cancer Research Center – Lyon

library_source: transcriptomic

data_processing:
Reads were aligned on the UCSC Homo sapiens hg19 genome using TopHat2.
Differential expression analysis was performed using Cuffdiff from Galaxy web site
Only genes with at least 1 read per million (FPKM) in at least 2 of the samples were kept for subsequent analyses.
Genome_build: hg19
Supplementary_files_format_and_content: Cuffdiff  txt files contain, FPKM,  log2 of the fold change, and Pvalue, and qvalue for each studied of conditions.

molecule: polyA RNA

organism: Homo sapiens

source_name: cell line

growth_protocol: HMLER cell lines were kindly provided by Anne-Pierre Morel (CRCL, Lyon, France). They were cultivated as previously described (Morel et al. PLoS One 2008). MDA-MB-231 cell lines were human breast cancer cell  line.

extract_protocol:
Total RNA was extracted with the NucleoSpin RNA kit (Macherey-Nagel, Hoerdt, France) following the manufacturer's instructions. RNA purity and integrity were examined on agarose gel, and quantified with a NanoDrop 1000 (Thermo Scientific, Wilmington, DE).
Illumina TruSeq RNA sample preparation Kit.

treatment_protocol: Cells were seeded at 1.105 per well in six-well plates. The day after plating, cells were  treated every day with 10 µM of 5-aza-2'-deoxycytidine (Sigma-Aldrich). Cells were collected 72 h after the start of the treatments.

tag: cell line

tag: treatment


title: 786O_VHL_H

city: Oxfors

first: James

last: Platt

address: Roosevelt Drive

country: United Kingdom

institute: University of Oxford

library_source: genomic

data_processing:
Reads were trimmed using trim galore and then in silico digested with DpnII using dpnII2E.pl (https://github.com/telenius/captureC/releases)
Resulting reads were then algined to genome using bowtie 1.0.0
Interaction frequencies were then determined using CCanalyser2.pl (https://github.com/telenius/captureC/releases)
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files contain interaction frequency from all sites

molecule: genomic DNA

source_name: Renal Carcinoma Cell Line

growth_protocol: Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin.

extract_protocol: Capture-C libraries were prepared as previously described (Davies et al. 2016 Nature Methods). Cells were fixed with 2% (vol/vol) formaldehyde for 10 min, quenched with 125 mM glycine in PBS and then lysed in cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, 0.2% Igepal, 1× cOmplete Protease Inhibitor Cocktail (Roche). Chromatin was digested with Dpn2 (New England Biolabs) at 37oC overnight.  Fragments were then diluted and ligated with T4 DNA ligase (Thermo Scientific) at 16oC overnight. Crosslinking was reversed by overnight incubation at 60oC with proteinase K (Bioline). The 3C libraries were then purified by phenol-chloroform and chloroform extraction followed by precipitation in ethanol at -80oC overnight. Digestion efficiency was determined by qPCR and gel electrophoresis. Sequencing libraries were prepared from 5 μg of each 3C library by sonication using a S220 focused-ultrasonicator (Covaris) to a average size of 200bp and indexed using NEBnext reagents (New England Bioloabs) according to protocol. Enrichment of 1-2 μg of indexed library incubated with 13 pmol of a pool of biotinylated oligonucleotides (Intergrated DNA technologies or Sigma) was performed using the SeqCap EZ system (#06953212001, Roche/Nimblegen) following the manufacturer’s instructions. Two rounds of capture employing 48-72 hr and 24 hr hybridizations respectively were used. Capture enrichment was determined by qPCR. Correct library size was confirmed using a Bioanalyzer DNA 1000 or Tapestation D1000 kit (Agilent), and DNA concentrations were determined using a Qubit 2.0 Fluorometer (ThermoFisher Scientific).

treatment_protocol: For hypoxia treatment cells, were incubated for 16 hr in an In Vivo2 Hypoxia Work station (Ruskinn Technology) at 0.5% oxygen.

value: Hypoxia

tag: cell line modification

value: stable WT VHL expression


title: MAC_light_MNase_rep2

city: Ann Arbor

state: Michigan

country: USA

institute: University of Michigan

description: Light MNase treatment replicate 2


title: a704.si.lats

first: Chinnaiyan

middle: M

last: Arul

address: 5309 Comprehensive Cancer Center, 1400 E. Medical Center Drive

department: Michigan Center for Translational Pathology

description:
LATS siRNA
SI_13590
RIN=10

data_processing:
Following sequencing and base-calling, RNA-seq data was aligned using STAR (2.4.0g1) to the GRCh38.p1 reference genome, using the “basic” version of gencode 22 to construct the splice junction database. Expression of protein-coding genes was quantified by counting the reads overlapping exons of annotated protein coding genes in strand-specific mode.
Genome_build: GRCh38.p1
Supplementary_files_format_and_content: text (tab delimited) file that contains serial number, HUGO gene symbol, followed by FPKM values in each experimental condition.

source_name: Kidney

growth_protocol: Caki-1, HK2 and A704 cells were purchased from ATCC and grown in McCoy, Keratinocyte and RPMI media supplemented with 10% FBS, penicillin, and streptomycin.

extract_protocol:
Total RNA was isolated using miRNA easy kit (Qiagen) following manufacturer's protocol.
RNA-Seq libraries were generated using KAPA Stranded RNA-Seq Library Preparation Kit (Kappa Biosystems) following the manufacturer's instruction

treatment_protocol: siRNAs were obtained from Ambion and transfected (25nM) using Lipofectamine RNAimax (Invitrogen).  For transfection 0.4 million cells were plated in a 6-well plate before transfection. 48 hrs after transfection cells were trypsinized, counted and replated for proliferation assay. Remaining cells were used for total RNA isolation.

tag: disease state

value: Cancer

tag: cell type

value: Epithelial

tag: si target

value: LATS


title: HMLER_Ctrl1

value: HMLER


title: MIC_heavy_MNase_rep2

description: Heavy MNase treatment replicate 2

source_name: micronucleus (MIC)


title: MAC_light_Mnase_rep1

description: Light MNase treatment replicate 1

value: sucrose gradient


title: HMLER_DAC_1


title: HMLER_DAC_2


title: 786O_VHL_N

value: Normoxia


title: MAC_heavy_MNase_rep1

description: Heavy MNase treatment replicate 1


title: ChIP_H3K4me2_Rep_2

data_processing:
Reads of each sample were firstly mapped to the MAC genome assembly (http://ciliate.org/index.php/home/downloads) of T. thermophila using TopHat2 allowing 2 edit distances (including mismatch and indel). Unique mapped reads were used for analysis.
Unique mapped reads were extracted, reads count of each called nucleosome are calculated using featureCounts software.
The mapped reads were visualized using Gbrowse2.
Genome_build: Tetrahymena thermophila MAC genome assembly (http://ciliate.org/index.php/home/downloads)
Supplementary_files_format_and_content: Reads counts of called nucleosomes

extract_protocol:
For native ChIP, 2x108 vegetative growing Tetrahymena cells of RPB7-CHA were collected at 350 g for 5 minutes. After cell lysis, nuclei were isolated by differential centrifugation following established protocols. MAC fractions containing less than 5% MIC (count/count) were used for MNase digestion. MNase digestion was carried out at room temperature for 15 min (50 mM Tris-HCl, 5 mM CaCl2, 1 mM β-mercaptoethanol, 0.1% NP-40, 0.1 mg/ml BSA, 2 U/μL MNase), stopped by adding 10 mM EGTA, 1 mM EDTA and 10 mM sodium butyric. Add equal volume of T350+Tx+Pi buffer (350 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) with PMSF and RNase A. After centrifuge, the supernatant was collected as input fraction. Anti-HA Magnetic Beads (Mouse) (Pierce #88836) was added to the input and incubated at 4oC with rotating for overnight. After wash with T150+Tx+Pi buffer (150 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) (three times) and T350+Tx+Pi buffer (350 mM Tris, 20 mM KCl, 2 mM MgCl2, 0.1% TritonX-100, Protease inhibitor cocktail) (one time), beads were eluted with 500 μL of HA peptide and DNA was recovered according to standard protocol. Recovered DNA product was sent to DNA sequencing core for CHIP-seq. For crosslink ChIP, 1x108 vegetative growing WT Tetrahymena cells were resuspended in 50 mL of fixation buffer (1% paraformaldehyde in 1x DPBS) and incubated at room temperature for 30 minutes. Cells were washed with ice-cold 1x DPBS for three times. Add 5 mL of SDS Lysis buffer and PMSF to the cell pellet and homogenize cells fully with Syringe passage followed by sonication (30s on, 30s off for 20 minutes as a cycle, five cycles in total). After centrifuge and filtration, the soluble fraction was recovered and diluted in the CHIP dilution buffer (1:5) as the input. 10 μg of α-H3K4me2 antibody (abcam 07-030) was added into the input fraction and incubated at 4oC overnight. On the second day, 50 μL of pre-washed Dynal magnetic beads was added into the input-antibody mixture and incubated at 4oC with rotating for 2 hours. After washing with Wash Buffer A (three times), RIPA Wash Buffer (three times), Last Wash Buffer (two times). Beads were eluted with 500 μL of SDS Elution Buffer and DNA was recovered according to standard protocol. Recovered DNA product was sent to DNA sequencing core for CHIP-seq.
Libraries were prepared with the Illumina TruSeq protocol according to the manufacturer’s protocol.

treatment_protocol: For Pol II occupancy, cells with HA-tagged RPB7 protein was used, and native ChIP performed using anti-HA Magnetic Beads (Mouse) (Pierce #88836). For H3K4me2 occupancy,wild type cells used, and crosslink ChIP performed using α-H3K4me2 antibody (abcam 07-030) .

tag: antibody

tag: antibody vendor/catalog


title: Input_H3K4me2_Rep_2


title: ChIP_H3K4me2_Rep_1


title: MDA_MB_231_bound_MeDP

data_processing:
Reads were aligned on the UCSC Homo sapiens hg19 genome using Bowtie (0.12.7).
Duplicate reads were filtered using SAMTools rmdup.
Bigwig files were obtained using Pipeline Fish the ChIPs: a pipeline for automated genomic annotation of ChIP-Seq data. Barozziet  al. Biology  Direct 2011,6:51.
Genome_build: hg19

extract_protocol:
Genomic DNA was sheared to a mean fragment length of 300-500 bp through sonication. Methylated-DNA precipitation (MeDP) was carried out using the MethylMiner Methylated DNA Enrichment Kit (Life Technologies) from 1µg of sheared DNA. Pools of 5 independent experiments, library preparation and high throughput sequencing (single-end 50 bp sequencing on Illumina HiSeq 2000) were performed at Beijing Genomics Institute (Hong-Kong, China).
DNA-end was repaired to overhang a 3'-dA, then adapters were ligated to the end DNA fragments. DNA fragments with proper size (usually 100-300bp, including adaptor sequence) were selected after PCR amplification.

treatment_protocol: No treatments.

tag: protocol

value: sonication,  MeDP


title: ChIP_RPB7_Rep_2

value: Pierce #88836


title: 48h_5FU_R2

city: Adelaide

first: David

middle: L.

last: Adelson

state: SA

address: North Terrace

department: Genetics & Evolution

country: Australia

institute: The University of Adelaide

description: strand-specific RNA-seq

data_processing:
Illumina Casava1.7 software used for basecalling.
Low quality and adaptor sequences in raw reads were trimmed using Trim_galore (v0.3.7, Babraham Bioinformatics) with the following parameters: -- stringency 6 --paired. Then cleaned reads were aligned to the reference genome (hg19, UCSC) using STAR_2.4.0j with following parameters: --outSAMstrandField intronMotif --outSAMattributes All --outFilterMismatchNmax 10 --seedSearchStartLmax 30.
Differential expression analysis was performed with edgeR and DE genes were selected with a False Discovery Rate (FDR) < 0.05.
Genome_build: hg19, UCSC
Supplementary_files_format_and_content: Tab delimited matrix showing raw counts of refGenes; GTF format including genomic coordinates of de novo identified lncRNAs;

molecule: total RNA

growth_protocol: MCF-7 cells were ordered from ATCC (HTB-22TM, Manassas, VA, USA). Cells were cultured in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 0.01 mg/mL human recombinant insulin (Thermo Fisher Scientific) at 37 °C with 5% CO2

extract_protocol:
Total RNA was isolated with the mirVana PARIS Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol.
Stranded mRNA libraries for Illumina sequencing were prepared using the NEBNext(r) Ultra Directional RNA kits from New England Biolabs, Inc. according to the manufacturer's protocol (Version 2.0 July 2013).  Actinomycin D was added during cDNA synthesis to ensure high levels of strand specificity.  All libraries were run on a Bionanalyzer to confirm library size and yield.  Barcoded libraries were normalized and pooled based on concentrations determined by qPCR with Library Quantification kits from KAPA Biosystems.

treatment_protocol: Drug treatments were designed as follows: CKI in 2 doses (1.0 mg/mL and 2.0 mg/mL) or 5-FU in one dose (10 ug/mL) for 24 and 48 hours

tag: tumor type

value: breast cancer


title: 48h_5FU_R1

description: strand-specific RNA-seq


title: 48h_2mg_R3

description: strand-specific RNA-seq


title: Input_H3K4me2_Rep_1


title: ChIP_RPB7_Rep_1


title: 48h_5FU_R3

description: strand-specific RNA-seq


title: Input_RPB7_Rep_2


title: HMLER_Ctrl2


title: HMLER_Input

value: sonication


title: Input_RPB7_Rep_1


title: 48h_2mg_R2

description: strand-specific RNA-seq


title: HMLER_Bound_MeDP

value: sonication, MeDP


title: 48h_2mg_R1

description: strand-specific RNA-seq


title: 48h_Ctrl_R3

description: strand-specific RNA-seq


title: 48h_1mg_R1

description: strand-specific RNA-seq


title: 48h_1mg_R2

description: strand-specific RNA-seq


title: 48h_Ctrl_R2

description: strand-specific RNA-seq


title: 48h_Ctrl_R1

description: strand-specific RNA-seq


title: MDA_MB_231-Input


title: Input-seq_in_wild-type_cells_replicate1 [part 3]

city: Umeå

first: Yuri

middle: B

last: Schwartz

address: Byggnad 6L, NUS

country: Sweden

institute: Umeå University

data_processing:
Base calling and quality score assignment were done with built-in Real-time analysis (RTA) software (Illumina).
ChIP-seq reads were aligned to the dm3 genome assembly using bowtie2 using the default parameters.
The SAM files with aligned reads were tested for strand cross-correlation using pyicos.
The SAM files were converted to wig format using pyicos. During conversion, the reads were extended based on the results of the above cross-correlation analysis.
Genome_build: dm3
Supplementary_files_format_and_content: wig files were generated using pyicos; Scores represent the number of reads overlapping each genomic position.

organism: Drosophila melanogaster

source_name: Cultured cells, WT, input

growth_protocol: Cells were grown to confluence in Schneider's medium (Lonza) supplemented with 10% FBS, 100U/ml of Penicillin G, 100ug/ml of Streptomycin sulfate.

extract_protocol:
Live cells were crosslinked with 1.8% formaldehyde for 10 min at 25C and soluble chromatin prepared as described in Schwartz et al., 2006. ChIP was done as described in Schwartz et al., 2006.
Libraries were prepared using NEBNext ChIP-Seq Library Prep Master Mix Set and Multiplex Oligos for Illumina (New England BioLabs) following the manufacturer instructions.

tag: mutation

tag: chip antibody


title: Input-seq_in_wild-type_cells_replicate2 [part 3]


title: Pc_ChIP-seq_in_wild-type_cells_replicate1 [part 3]

source_name: Cultured cells, WT, Pc ChIP

value: Pc from Poux et al., 2001


title: Pc_ChIP-seq_in_wild-type_cells_replicate2 [part 3]


title: 24h_5FU_R2

description: strand-specific RNA-seq


title: Input-seq_in_Su(z)12-minus_cells_replicate2 [part 3]

source_name: Cultured cells, Su(z)12-null, input


title: 24h_5FU_R1

description: strand-specific RNA-seq


title: Pc_ChIP-seq_in_Su(z)12-minus_cells_replicate1 [part 3]

source_name: Cultured cells, Su(z)12-null, Pc ChIP


title: 24h_2mg_R4

description: strand-specific RNA-seq


title: Pc_ChIP-seq_in_Su(z)12-minus_cells_replicate2 [part 3]


title: 24h_2mg_R3

description: strand-specific RNA-seq


title: 24h_2mg_R2

description: strand-specific RNA-seq


title: Psc_ChIP-seq_in_wild-type_cells_replicate2 [part 2]

data_processing:
Base calling and quality score assignment were done with built in Real-time analysis (RTA) software (Illumina)
ChIP-seq reads were aligned to the dm3 genome assembly using bowtie2 using the default parameters
The SAM files with aligned reads were tested for strand cross-correlation using pyicos
The SAM files were converted to wig format using pyicos. During conversion the reads were extended based on the results of the above cross-correlation analysis
Genome_build: dm3
Supplementary_files_format_and_content: wig files were generated using pyicos; Scores represent the number of reads overlapping each genomic position.

source_name: Cultured cells

growth_protocol: Cells were grown to confluence in Schneider's medium (Lonza) supplemented with 10% FBS, 100U/ml of Penicillin G, 100ug/ml of Streptomycin sulfate

value: Psc from Poux et al., 2001


title: Input-seq_in_wild-type_cells_replicate1 [part 2]


title: Psc_ChIP-seq_in_wild-type_cells_replicate1 [part 2]


title: Input-seq_in_wild-type_cells_replicate2 [part 2]


title: Input-seq_in_Su(z)12-minus_cells_replicate2 [part 2]


title: Psc_ChIP-seq_in_Su(z)12-minus_cells_replicate2 [part 2]


title: Input-seq_in_Su(z)12-minus_cells_replicate1 [part 2]


title: 48h_1mg_R3

description: strand-specific RNA-seq


title: 24h_1mg_R4

description: strand-specific RNA-seq


title: 24h_5FU_R3

description: strand-specific RNA-seq


title: 24h_1mg_R2

description: strand-specific RNA-seq


title: Psc_ChIP-seq_in_Su(z)12-minus_cells_replicate1 [part 2]


title: Input-seq_in_wild-type_cells_replicate1 [part 1]


title: E(z)_ChIP-seq_in_wild-type_cells_replicate2 [part 1]

value: E(Z) from Kahn et al., 2006


title: MIC_heavy_MNase_rep1

description: Heavy MNase treatment replicate 1

value: gel electrophoresis


title: 24h_Ctrl_R2

description: strand-specific RNA-seq


title: E(z)_ChIP-seq_in_wild-type_cells_replicate1 [part 1]


title: 24h_Ctrl_R3

description: strand-specific RNA-seq


title: E(z)_ChIP-seq_in_Psc-minus_cells_replicate2 [part 1]


title: Input-seq_in_wild-type_cells_replicate2 [part 1]


title: 24h_1mg_R1

description: strand-specific RNA-seq


title: Input-seq_in_Psc-minus_cells_replicate2 [part 1]


title: Input-seq_in_Psc-minus_cells_replicate1 [part 1]


title: Input-seq_in_Su(z)12-minus_cells_replicate1 [part 3]


title: 0h_Ctrl_R4

description: strand-specific RNA-seq


title: 24h_Ctrl_R1

description: strand-specific RNA-seq


title: 0h_Ctrl_R5

description: strand-specific RNA-seq


title: E(z)_ChIP-seq_in_Psc-minus_cells_replicate1 [part 1]


title: FabT_40_ST_3

city: Houston

first: Stephen

middle: B.

last: Beres

state: TX

address: 6670 Bertner Ave.

department: Pathology and Genomic Medicine

institute: Houston Methodist Research Institute

data_processing:
multiplexed SE libraries were sequenced on an Illumina HiSeq 2500 instrument, basecalling and demultiplexing was done on the instrument, and the RNA-Seq data was output as fastq files
RNA-Seq reads were mapped to reference strain MGAS5005 (Genbank accession number NC_007297) using CLC Genomics Workbench 8.0.
mapping reads from each strain to the reference using CLC Genomics Workbench 8.0 yields raw and normalized (RPKM) expression values for each gene.
genes encoding ribosomal RNAs and tRNAs were filtered out prior to running differential expression analyses
differential expression analysis was perfomed using DESeq2 v1.6.3 runing in R version 3.1.3. Raw reads were used as the input and normalized expression generated using DESeq2's default normalization method Relative log expression (RLE) approcah was used to generate fold change values for each gene. Multiple testing correction was performed using Bonferroni correction method.

organism: Streptococcus pyogenes

source_name: M1GAS2221ΔfabT_Early stationary phase

growth_protocol: cells were grown in Todd-Hewitt broth supplemented with yeast extract (THY media) at 35C and 40C in atmosphere supplemented with 5% CO2

extract_protocol:
pelleted cells were mechanically disrupted using silica beads, RNA was isolated using RNeasy kit, contaminating DNA was degraded using Turbo DNA-free (Ambion), and rRNA was depleted using Ribo-Zero (Epicentre)
resultant RNA was reverse transcribed to cDNA and sequencing libraries were generated using ScriptSeq RNA-seq preparation kit (Epicentre)

treatment_protocol: at the desired growth stage, mid-exponential OD 600nm 1.0 or early-stationary (2 hours post exponential growth phase), the culture was treated with RNAprotect Bacteria Reagent and cells were harvested by centrifugation

tag: serotype

tag: strain

tag: genotype/variation

value: Isogenic fabT deletion mutant

tag: growth phase/temperature

value: Early stationary phase (SP, OD600nm=1.5). 40-degree growth

tag: sample type

value: cell pellet


title: FabT_40_ST_1


title: 0h_Ctrl_R2

description: strand-specific RNA-seq


title: FabT_40_ST_2


title: WT2221_40_ST_3

source_name: M1GAS2221_Early stationary phase

value: wild type


title: WT2221_40_ST_1


title: 786O_IN_2

description: sonicated DNA not ChIPed

data_processing:
Illumina adaptor sequences were trimmed from the ChIP-seq reads using Trimgalore (0.3.3) and the reads were then aligned to Genome Reference Consortium GRCh37 (hg19) using BWA (0.7.5a-r405)
Low quality mapping was removed (MapQ < 15) using SAMtools (0.1.19) and reads mapping within the Duke Encode black list regions (http://hgwdev.cse.ucsc.edu/cgi-bin/hgFileUi?db=hg19&g=wgEncodeMapability) were excluded using BEDTools (2.17.0)
Duplicate reads were marked using Picard tools (1.106) (http://picard.sourceforge.net/) and BigWig files generated for visualization
peaks were identified using T-PIC (Tree shape Peak Identification for ChIP-Seq) and MACS (Model-based analysis of ChIP-Seq)
Genome_build: hg19
Supplementary_files_format_and_content: bed file containing peak calls

source_name: Renal Cancer Cell line

extract_protocol:
ChIP-seq: Cells were fixed with 1% formaldehyde and quenched with glycine (125mM). After washing, cells were lysed (0.5%SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and sonicated (Diagenode Biorupter, Belgium). Chromatin was immunoprecipitated using antibodies indicated. Eluted chromatin was heated to reverse cross-linking, treated with proteinase-K, RNaseA and purified on a qiagen PCR pufircation column.
Libraries were prepared using PrepX Complete ILMN DNA library kit (#400076, Wafergen) by the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics, Oxford, UK.

tag: cell line

tag: repeat

value: INPUT DNA


title: 786O_RNApol2_1

tag: treatment

value: RNApol2 (#sc-899, Santa Cruz )


title: WT2221_40_ST_2


title: 786O_IN_1

description: sonicated DNA not ChIPed


title: 786O_RNApol2_2


title: FabT_35_ST_3

value: Early stationary phase (SP, OD600nm=1.5). 35-degree growth


title: 786O_CTCF_2

value: Normoxia

value: CTCF (#07-729, Millipore)


title: FabT_35_ST_2


title: 786O_CTCF_1


title: FabT_35_ST_1


title: WT2221_35_ST_3


title: 16 hpl Replicate 2

first: Pamela

middle: A

last: Raymond

state: MI

address: 830 N. University

department: Molecular, Cellular, and Developmental Biology

description:
DifferentialExpressionAnalysis.xlsx
RSEMGeneCounts.txt
RSEMNormFPKM.txt

data_processing:
Basecalls and quality scores performed using RTA 1.13.48 and Fastq files generated from reads passing chastity filtering.
Trim Galore! (v0.2.7) (Krueger, 2012) was used to trim adapter sequences and poor quality bases (below Phred of 20) from the reads while removing any reads that were less than 20 nucleotides long, using the default parameters. Trim Galore! makes use of cutadapt (v1.4.2) (Martin, 2011) (-f fastq -e 0.1 -q 20 -O 1 -a AGATCGGAAGAGC file.fq.gz) . The quality of the reads was assessed before and after trimming with FastQC (v0.10.1) (Andrews, 2010).
Quality trimmed and filtered reads were aligned to release 78 of the Zv9 Ensembl genome build with bowtie2 (v2.2.6) (Langmead and Salzberg, 2012) and gene-level quantitation was performed with RSEM (v1.2.22) (Li and Dewey, 2011). This was done using the rsem-calculate-expression command from RSEM, which calls bowtie2 (--sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1) and streams reads into RSEM for quantitation.
The gene-level counts output from RSEM were filtered to remove noise prior to normalization with trimmed means of M (TMM), such that only genes with a FPKM (fragments per kilobase of exon per million reads mapped) value greater than 1 in all replicates of any time-point were retained. Counts per million (CPM) were determined using edgeR (v3.10.2) (Robinson et al., 2010), genes with a CPM < 1 in all samples were removed, and remaining counts were TMM normalized. Multidimensional scaling analysis with edgeR showed that one of the replicates at each of the 8 and 16 hpl timepoints did not cluster with the others. Limma (v3.24.15) was used to voom transform the filtered count data by empirically deriving and applying quality weights to the samples (Law et al., 2014; Ritchie et al., 2015). These weighted values were used to calculate differential expression using limma. Annotations for each gene were added using biomaRt (v2.24.0) (Durinck et al., 2005; Kersey et al., 2014), including both the Danio rerio Entrez gene identifiers and the corresponding Mus musculus Entrez orthologous gene identifiers.
Genome_build: Zv9 (Release 78)
Supplementary_files_format_and_content: Excel file containing differential expression data, or tab-delimited text files containing count and FPKM data.

organism: Danio rerio

source_name: Müller glia

extract_protocol:
Retinas were removed and dissociated to obtain single-cell suspensions. Müller glia were collected with fluorescently-actived cell-sorting. RNA was isolated with TRIzol LS according to recommenders protocol. RNA quality was checked via BioAnalyzer and samples with an RNA integrity number (RIN) > 7.0 were used for RNA-seq library construction.
Stranded RNA-seq libraries were constructed using a modification of the Illumina TruSeq mRNA (Brooks, et al. Meth Mol Biol 2012). Each library was single-end sequenced in an independent lane of a GAIIx. Fastq files were generated from reads passing chastity filter.

treatment_protocol: Free-swimming fish exposed to intense light (>120,000 lux) for 30 minutes. Retinas harvested at 16 hours post-lesion (hpl).

tag: line

tag: tissue

value: neural retina

value: Muller glia

tag: age

value: 8-11 months

value: Light lesion, 16 hours post-lesion


title: 16 hpl Replicate 3

tag: cell type


title: 786O_H3K27ac_2

value: H3K27ac (#ab4729, Abcam)


title: 786O_H3K27ac_1


title: WT2221_35_ST_2


title: 16 hpl Replicate 1


title: 8 hpl Replicate 3

treatment_protocol: Free-swimming fish exposed to intense light (>120,000 lux) for 30 minutes. Retinas harvested at 8 hours post-lesion (hpl).

value: Light lesion, 8 hours post-lesion


title: 786O_H3K4me3_1

value: H3K4me3 (#9751, Cell Signaling Technology)


title: 786O_H3K4me3_2


title: 786O_H3K4me1_2

value: H3K4me1 (Millipore, #07-436)


title: WT2221_35_ST_1


title: 8 hpl Replicate 2


title: FabT_40_ME_3

value: Isogenic fabT deletion mutant

value: Mid-exponential (ME, OD600nm=1.0). 40-degree growth


title: 8 hpl Replicate 1


title: FabT_40_ME_2

value: cell pellet


title: 0 hpl Replicate 3

value: Control, unlesioned


title: MCF7_IN_H

description: sonicated DNA not ChIPed

source_name: Breast Cancer Cell  Line

value: Hypoxia

value: INPUT DNA


title: 0 hpl Replicate 2


title: MCF7_IN_N

description: sonicated DNA not ChIPed


title: 786O_H3K4me1_1


title: FabT_40_ME_1


title: WT2221_40_ME_3

value: wild type


title: FabT_35_ME_3

value: Mid-exponential (ME, OD600nm=1.0). 35-degree growth


title: MCF7_CTCF_H2

value: CTCF (#07-729, Millipore)


title: WT2221_40_ME_1


title: FabT_35_ME_2


title: FabT_35_ME_1


title: WT2221_35_ME_3


title: WT2221_35_ME_2


title: MCF7_CTCF_H1


title: MCF7_H3K27ac_H2


title: MCF7_CTCF_N2

value: Normoxia


title: MCF7_H3K27ac_N1


title: MCF7_H3K4me1_H2


title: 0 hpl Replicate 1

value: neural retina

value: Muller glia


title: Sample_Os_5D_RNA_Seq

city: Shanghai

first: Lin

last: XU

address: 300 Fenglin Road

department: Chinese Academy of Sciences

country: China

institute: Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences

description: sample 3

data_processing:
The sequencing reads were cleaned, including removing bases with low quality score (<20) and irregular GC content based on FastQC, cutting sequencing adaptor followed by filtering short reads.
The cleaned reads were mapped to Oryza Sativa genome (msu7) using BOWTIE2.2.9 for DNA sequencing and TOPHATv2.0.8 for RNA sequencing, both with default settings.
The duplicated reads were removed, and only alignments with MAPQ score > 20 were kept for further analysis.
HTSeq-count was used to preprocess the RNA-seq alignments and deseq was used to detect the differentially expressed genes.
Genome_build: MSU7
Supplementary_files_format_and_content: read counts for genes(msu7) was provided. The format is tab delimited file format. Each row contains two columns: the first is the feature, and the second is the count of reads which are mapped to the feature.

organism: Oryza sativa Japonica Group

source_name: base region（2 mm) of 7 mm rice leaf explants at time 5 DAC on Callus Induced Medium

growth_protocol: Rice was grown at 29ºC with a 12-h light (~ 80000 Lux) and 12-h dark photoperiod in the greenhouse or plant chamber.

extract_protocol:
RNA extraction using Trizol protocols.
Library construction and deep sequencing were performed by Genergy Biotechnology Co. Ltd. (Shanghai, China) using Illumina HiSeq 3000 following the manufacturer’s instructions (Illumina)

treatment_protocol: The rice leaf was grown at 0, 2 and 5 days on Callus Induced Medium.

value: callus


title: WT2221_40_ME_2


title: MCF7_H3K4me1_H1


title: Sample_Os_2D_RNA_Seq

description: sample 2

source_name: base region（2 mm) of 7 mm rice leaf explants at time 2 DAC on Callus Induced Medium


title: Sample_Os_0h_RNA_Seq

description: sample 1

source_name: base region（2 mm) of 7 mm rice leaf explants at time 0 DAC on Callus Induced Medium

value: base region of young leaf


title: MCF7_H1

tag: cell line modification


title: MCF7_H2


title: MCF7_CTCF_N1


title: MCF7_N2


title: MCF7_N1


title: MCF7_H3K4me1_N2


title: MCF7_H3K4me1_N1


title: MCF7_H3K27ac_H1

value: H3K27ac (#ab4729, Abcam)


title: KO3

city: shanghai

first: zheng

middle: yan

last: sen

state: shanghai

address: NO.500 dong chuan road

institute: ECNU

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: mm8
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

organism: Mus musculus

source_name: mice colorectal cancer tissues

growth_protocol: generated Gprc5a knockout mice by homologous recombination

extract_protocol:
Total RNA was isolated from the colorectal cancer tissues using the RNAeasy Mini Kit (Qiagen, Valencia, CA). Total RNA prepared was submitted to Shanghai Personal Biotechnology Co., Ltd, where RNA intergrity was confirmed using the Illumina HiSeq 2000 system.
mRNA was purified from total RNA by oligo-dT magnetic beads and normalized to 150 bp short fragments. Double-strand cDNA libraries were prepared and constructed using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA). The synthesized cDNA libraries were diluted into 4-5 nM and sequenced.

tag: tissue type

value: tumor

tag: genotype

value: Gprc5a knockout


title: KO1


title: WT3

value: WT


title: KO2


title: MCF7_H3K27ac_N2


title: WT2221_35_ME_1


title: WT2


title: 786O_2

source_name: Renal Carcinoma Cell Line


title: 03-1-0185-1

status: Public on Sep 13 2016

city: Minneapolis

first: Casey

last: Dorr

state: MN

address: 701 Park Avenue

institute: Minneapolis Medical Research Foundation

data_processing:
Illumina Native files converted to FASTQ by Minnesota Supercomputing Institute
FASTQ files assessed for quality by FastQC:Read QC
Paired-end reads aligned to human genome (GRCH37/hg19) via Tophat2 using iGenome UCSC reference annontation
Transcript assembly and abundance was determined using Cufflinks with hg_19_genes_2012-30-09.gtf reference annotation
Genome_build: GRCH37/hg19

source_name: blood

growth_protocol: Adults receiving living donor kidney allografts were studied. Patients received thymoglobulin induction and maintenance therapy with tacrolimus or cyclosporine, with mycophenolate and short course steroids to days 5–7 post-transplant. Four of the patients received tacrolimus or cyclosporine prior to transplantation. Five patients were receiving steroids and 9 were receiving mycophenolate at baseline for underlying disease. The subjects had no rejection or any previous rejection at time of each sample collection. Sequential whole blood samples for isolation of PBMCs were collected at baseline (pre-transplant, n = 32), week 1 (n = 31), month 3 (n = 18) and month 6 (n = 15) post-transplant. Some samples were not obtained because patients did not return to the transplant clinic for follow-up center visits and clinical follow ups were performed by referring physician. All patients in this study provided written informed consent following protocol that was approved by the institutional review board of the University of Minnesota.

extract_protocol:
Qiagen QIAamp RNA Blood Mini kit (Germantown, MD)
RNA was quantitated with a Nanodrop 800 spectrophotometer. One μg of each RNA sample was used to prepare RNAseq libraries based on the method as outlined by Zhong and colleagues [reference 17 in published manuscript] with modifications and added quality checks. Sample quantity and integrity was checked using RiboGreen analysis and an Agilent 2100 Bioanalyzer or Caliper equivalent. Each sample passing quality control (RNA mass > 1 μg and RNA Integrity Number > 6) was used to create a polyA+ stranded barcoded RNAseq library using standard protocols. Five samples were pooled for each lane for Illumina Hi-seq 2000 sequencing to generate 20–40 million mapped paired-end reads per sample.

treatment_protocol: Blood was collected into BD Vacutainer EDTA coated tubes. RNA was isolated from approximately 12 mLs of whole blood PBMCs using the Qiagen QIAamp RNA Blood Mini kit (Germantown, MD) within 2 hours of blood draw.

tag: time of blood draw

value: 1 week post-transplant

tag: race

value: Caucasian / White

tag: ethnicity

value: Non-Hispanic/Non-Latino

tag: gender

value: M

tag: primary disease

value: Diabetes Type 1

tag: transplanted organ

value: Kidney

tag: transplant type

value: kidney

tag: age at transplant


title: 03-1-0196-0

value: pre-transplant

value: Polycystic


title: 03-1-0196-1


title: 786O_1

tag: cell line

value: Normoxia


title: 03-1-0185-0


title: 03-1-0180-1

value: Glom-sclerosis,chr&unspecified


title: 03-1-0180-0


title: 03-1-0149-0

value: American Indian orAlaskaNative

value: Unknown

value: Diabetes Type 2


title: 03-1-0174-1

value: FSGS-Focal sclerosing GN


title: 03-1-0174-0


title: 03-1-0149-3

value: 3 months post-transplant


title: 03-1-0149-1


title: 03-1-0138-6

value: 6 months post-transplant

value: F


title: 03-1-0148-3


title: 03-1-0138-0

value: Caucasian / White


title: 03-1-0138-3

value: Non-Hispanic/Non-Latino


title: 03-1-0137-3

value: Black or African American

value: Hypertension, nephrosclerosis


title: 03-1-0137-1

value: 1 week post-transplant

value: Kidney


title: 03-1-0137-0

value: pre-transplant

value: kidney


title: 03-1-0136-6


title: WT1

tag: strain

value: tumor

value: WT


title: 03-1-0125-3

value: American Indian orAlaskaNative

value: MPGN-membranoproliferative?typ


title: 03-1-0125-1

value: Unknown


title: 03-1-0136-1

value: Glom-sclerosis,chr&unspecified


title: 03-1-0171-3

value: 3 months post-transplant

value: M

value: GN-other chronicGlom.nephritis


title: 03-1-0125-0

value: F


title: 03-1-0171-0

value: Caucasian / White

value: Non-Hispanic/Non-Latino


title: 03-1-0116-6

value: 6 months post-transplant


title: 03-1-0148-0

value: Diabetes Type 2


title: 03-1-0116-0

value: Kidney


title: 03-1-0113-1

value: 1 week post-transplant

value: Polycystic


title: 03-1-0116-1

value: kidney


title: 03-1-0113-6


title: 03-1-0148-1


title: 03-1-0101-0

value: pre-transplant

value: Alport's/Famil Hered neph


title: 03-1-0096-1


title: 03-1-0096-6


title: 03-1-0093-1


title: 03-1-0093-6


title: 03-1-0093-0


title: 03-1-0093-3


title: 03-1-0113-0


title: 03-1-0092-1

value: Black or African American

value: Hypertension, nephrosclerosis


title: 03-1-0092-6

value: Unknown


title: 03-1-0092-0


title: 03-1-0088-3

value: 3 months post-transplant


title: 03-1-0088-1

value: M


title: 03-1-0083-0


title: 03-1-0083-1


title: 03-1-0083-3


title: 03-1-0082-0

value: GN-membranous glom.nephritis


title: 03-1-0082-1


title: 03-1-0096-0


title: 03-1-0071-6

value: F

value: Diabetes Type 1


title: 03-1-0071-3

value: Caucasian / White

value: Non-Hispanic/Non-Latino


title: 03-1-0101-1


title: 03-1-0071-1


title: 03-1-0071-0


title: 03-1-0066-1

value: Nephropathy, IgA (Bergers)


title: 03-1-0066-3

value: Kidney


title: 03-1-0065-1

value: 1 week post-transplant

value: Thin basement membrane dz


title: 03-1-0065-6

value: 6 months post-transplant

value: kidney


title: 03-1-0065-0

value: pre-transplant


title: 03-1-0066-0


title: 03-1-0059-0

value: simultaneous pancreas and kidney


title: 03-1-0059-1


title: 03-1-0058-0

value: Cystinosis


title: 03-1-0058-1


title: 03-1-0051-1


title: 03-1-0083-6

value: Polycystic


title: 03-1-0038-3

value: 3 months post-transplant

value: Wegener's Granulomatosis


title: 03-1-0038-1

value: M


title: 03-1-0036-6

value: FSGS-Focal sclerosing GN


title: 03-1-0036-3


title: 03-1-0038-0


title: 03-1-0033-1

value: Unknown


title: 03-1-0036-1

value: Caucasian / White


title: 03-1-0036-0

value: Non-Hispanic/Non-Latino


title: 03-1-0028-0


title: 03-1-0033-0


title: 03-1-0027-0


title: 03-1-0027-1


title: 03-1-0028-1


title: 03-1-0058-6

value: F


title: 03-1-0058-3

value: Kidney


title: 03-1-0051-6

value: Diabetes Type 1


title: 03-1-0026-1

value: 1 week post-transplant

value: Nephritis,@2 other drugs


title: 03-1-0026-6

value: 6 months post-transplant

value: kidney


title: 03-1-0026-0

value: pre-transplant


title: 03-1-0022-3

value: Lupus (SLE)


title: 03-1-0006-6

value: Hypertension, nephrosclerosis


title: 03-1-0026-3

value: 3 months post-transplant


title: 03-1-0022-0


title: 03-1-0006-0


title: 03-1-0051-3


title: 03-1-0022-1


title: U2AF65-knockdown MCF-7 cells

city: Beijing

first: Mai

last: Sanyue

address: 27 Taiping Rd, Haidian District

institute: Beijing Institute of Biotechnology

description: All_Fragments.xls and All_UQFPKM.xls

data_processing:
Raw reads were discarded if they contained more than 2-N bases. A FASTX-Toolkit (Version 0.0.13) was used to obtain clean reads, and the raw sequencing data were also evaluated using FAST-QC.
The splicing-junction mapper from RNA-Seq TopHat (v2.0.6) was employed for RNA-seq alignment. Based on gene annotation of the genome, aligned reads with more than one genome location were discarded due to their ambiguous locations.
Uniquely localized reads were used to calculate the read number and the reads per kilobase and per million reads (RPKM) value for each gene.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

source_name: MCF-7 cells

growth_protocol: wild-type and RBM39-knockdown (or U2AF65-knockdown) MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (GIBCO Thermo Fisher) containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin.

extract_protocol:
10 μg of total RNA was treated with 1 U RQ1 DNase (Promega) to remove residual DNA. 10 μg of total RNA was used for RNA-Seq library preparation.
Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Thermo Fisher). Prior to directional RNA-seq library preparation, the purified mRNAs were fragmented with ion at 95°C, followed by end repair and 5' adaptor ligation. Reverse transcription was then performed with primers harboring a 3' adaptor sequence and randomized hexamers. cDNA was purified and amplified, and PCR products corresponding to 200-500 bp were purified, quantified and stored at -80°C until sequencing.

treatment_protocol: Upon reaching 60-80% confluence, the cells were transfected with 30 pmol of siRNA (GenePharma) using Lipofectamine RNAiMAX (Thermo Fisher). The cells were incubated for 2 days at 37°C following transfection, after which total RNA was extracted.

value: U2AF65-knockdown MCF-7 cells

value: human breast adenocarcinoma


title: 03-1-0051-0

value: M


title: RBM39-knockdown MCF-7 cells

description: All_Fragments.xls and All_UQFPKM.xls

value: RBM39-knockdown MCF-7 cells


title: HCT116WT-treated NOMe-seq

city: Augusta

first: Huidong

last: Shi

state: GA

department: Georgia Cancer Center

institute: Augusta University

description: Chromatin accessibility and DNA methylation analysis using targeted bisulfite sequencing

data_processing:
Library strategy: Targeted DNA capture followed by bisulfite treatment
Alignment: The raw bisulfite sequencing reads were first converted to “unmethylated reads” via the conversion of all Cs to Ts in silico. The unmethylated, converted sequencing reads were then aligned to the two copies of in silico converted human reference genome (hg19) using BWA. The results of both mappings were combined, and any reads mapped against both copies of converted reference were resolved by identifying their strand based on the TCAG ratio and the mapping positions used accordingly.
Methylation calculation: The methylation level [(# of methylated)/(# of methylated + # of unmethylated)] was calculated after measuring the amount of methylation (C->C) and unmethylation (C->T) in the C position.
HCT116WT-treated.reads.bed.gz: BED for the mapping position of reads
HCT116WT-treated.hcg.bed.gz: BED for the methylation level at HCGs
HCT116WT-treated.gch.bed.gz: BED for the methylation level at GCHs
Genome Build: hg19

source_name: Colon cancer cell line HCT116 treated with GpC methyltransferase (M.CviPI)

growth_protocol: HCT116  cells  were cultured at 37 C in McCoy’s 5a Medium supplementary with 10% FBS.

extract_protocol: The construction of methyl-seq library was carried out according to the protocol provided by Agilant. 1ug of genomic DNA is shared to around 125 ~ 175bp using a Covaris sonicator. The sheared DNA was end-repaired and ligated with methylated adapters. 500ng of the library DNA was hybridized with biotinylated oligo probes to capture single strand target DNA on the magnetic beads. The captured DNA was converted by bisulfite treatement and purified. The library was firstly amplified by 8 cycles of PCR. After purification, this PCR product was further amplified by 6 cycles of PCR and sequenced using illumina Hi-seq2000.

treatment_protocol: 1 million HCT16  cells were lysed to isolate nuclei. Then, the nuclei were treated with 100U of GpC methyltransferase (M.CviPI) in the presence of SAM for 10minutes at 37 C. Genomic DNA was isolated from the M.CviPI treated and untreated nuclei.

value: colon tumor

value: Epithelial cells


title: wild-type MCF-7 cells

description: All_Fragments.xls and All_UQFPKM.xls

value: wild-type MCF-7 cells


title: HCT116WT-untreated BS-seq

data_processing:
Library strategy: Targeted DNA capture followed by bisulfite treatment
Alignment: The raw bisulfite sequencing reads were first converted to “unmethylated reads” via the conversion of all Cs to Ts in silico. The unmethylated, converted sequencing reads were then aligned to the two copies of in silico converted human reference genome (hg19) using BWA. The results of both mappings were combined, and any reads mapped against both copies of converted reference were resolved by identifying their strand based on the TCAG ratio and the mapping positions used accordingly.
Methylation calculation: The methylation level [(# of methylated)/(# of methylated + # of unmethylated)] was calculated after measuring the amount of methylation (C->C) and unmethylation (C->T) in the C position.
HCT116WT-untreated.reads.bed.gz: BED for the mapping position of reads
HCT116WT-untreated.hcg.bed.gz: BED for the methylation level at HCGs
HCT116WT-untreated.gch.bed.gz: BED for the methylation level at GCHs
Genome Build: hg19

source_name: Colon cancer cell line HCT116


title: Trilostane+Fsh_6

city: Bergen

first: Diego

last: Crespo

address: Nordnesgaten 50

department: Reproduction and Developmental Biology

country: Norway

institute: Institute of Marine Research

data_processing:
Image analysis and base calling were done by the Illumina pipeline (CASAVA software v1.8)
The read counts were extracted using the Python package HTSeq, and normalized using the totally mapped reads per library
Sequenced reads were trimmed for adaptor sequence and then mapped to the zebrafish genome (Zv9) using STAR v2.3 (standard parameters for single-end reads)
Differential expression analysis was performed using the R/Bioconductor package NOISeq
Genome_build: Zv9
Supplementary_files_format_and_content: Tab-delimited text files include gene ID and raw counts for each sample.

source_name: Testis

extract_protocol:
Total RNA was isolated from testis tissue using the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA integrity was checked with an Agilent Bio-analyzer 2100 total RNA Nano series II chip (Agilent)
Illumina RNAseq libraries were prepared from 2 µg total RNA using the Illumina TruSeq RNA Sample Prep Kit v2 (Illumina, Inc.) according to the manufacturer’s instructions

value: testis

value: Adult

value: Wild type

tag: incubation

value: 48 hours


title: Trilostane+Fsh_5

tag: tissue


title: Trilostane+Fsh_3

tag: age


title: Trilostane+Fsh_4


title: Trilostane_6


title: Trilostane+Fsh_1


title: RNase H (HeLa)

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

data_processing:
Library strategy: 2OMe-seq
Raw reads were converted to FastQ, and examined using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For each run, the first 5 nucleotides at 5’-end were trimmed using the fastx_trimmer tool of the FASTX-Toolkit suite. Reads were then clipped from 3’ adapter sequences (requiring a minimum match of 10 nucleotides), using the fastx_clipper tool of the FASTX-Toolkit suite, discarding reads shorter than 25 nucleotides  (parameters: -a TGGAATTCTCGGGTGCCAAGG -l 25 -M 10 -Q 33; -a AGATCGGAAGAGCACACGTCT was replaced for Alkaline hydrolysis and 2OMe-seq Method #2 libraries, due to the different 3’ adapter used). Reads were mapped to the reference transcriptome, composed of 28S and 18S rRNA sequences, plus sequences of the 3 in vitro generated spike-ins (where included), using Bowtie v1.1.1 (parameters: -q -n 2 --norc -m 1 --best --strata).
Genome_build: Reads were mapped only to 18S and 28S rRNA sequence for each species.  Accessions are as follows: Homo sapiens 18S (NR_003286), Homo sapiens 28S (NR_003287), Mus musculus 18S (NR_003278), Mus musculus 28S (NR_003279).
Supplementary_files_format_and_content: Processed data file in BEDGraph format are provided (Columns: Transcript ID, Position, Position +1, Number of reads mapping 1nt downstream).

source_name: HeLa S3

growth_protocol: HeLa S3 cells were obtained from ATCC (cat. CCL2.2), and cultured in DMEM (4.5g/L D-Glucose), supplemented with 10% FBS, 0.1mM NEAA, 25U/ml penicillin, and 25 μg/ml streptomycin.

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
Briefly, 2 μg of total RNA and 1 μl of a 10μM random pentadecamers (NNNNNNNNNNNNNNN) mixture were heated 1 minute at 95°C, and annealed by slowly decreasing the temperature to 25°C. Reverse transcription was performed using 200U of SuperScript III Reverse Transcriptase (Invitrogen) by incubating at 50°C for 50 minutes, followed by 15 minutes at 75°C to inactivate the enzyme. Reaction volume (20 μl) was then brought to 100 μl with RNase H Digestion Buffer [20mM HEPES pH 8.0, 50mM KCl, 4mM MgCl2, 1mM DTT, 50 μg/ml BSA], supplemented with 20U Ribonuclease H (Ambion), and incubated for 1 hour at 37°C. DNA was degraded by adding 50 U of DNase I (Thermo Scientific) for 15 minutes at 37°C, and RNA was recovered by TRIzol (Invitrogen) extraction. 5’-P RNA fragments derived from RNase H digestion were ligated to the Illumina TruSeq RNA 5’ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) using 20U of T4 RNA Ligase 1 (NEB) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The adapter-ligated RNA was recovered by ethanol precipitation, and subjected to reverse transcription using 200U of SuperScript III Reverse Transcriptase, and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). The reverse transcription reaction was conducted in 50 minutes at 50°C, followed by 15 minutes at 75°C to inactivate the reverse transcriptase. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs recovery, and excess adapter removal was performed using Agencourt Ampure XP beads (Beckman Coulter). Following cleanup, cDNAs were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 80-200nt fragments was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, the adapter-ligated DNA was subjected to 15 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.

value: HeLa S3

tag: cell type

value: cervical adenocarcinoma


title: Trilostane+Fsh_2


title: Trilostane_3


title: Trilostane_4


title: 03-1-0006-3

value: Caucasian / White

value: Non-Hispanic/Non-Latino

value: Kidney

value: kidney


title: Trilostane_2


title: Trilostane_5


title: Trilostane_1


title: BC17 K1

city: Chilly Mazarin

first: Veeranagouda

last: Yaligara

address: Sanofi R&D

institute: Sanofi

data_processing:
FASTQ files were analyzed using Array studio V.8.0.3.67 (OmicSoft Corp). In the preprocessing  step, reads with a length below 18 nucleotides and average quality score below 20 were removed from FASTQ files
Genome_build: M. fascicularis miRNA reference sequences (generated in this study) were used for mapping
Supplementary_files_format_and_content: Excel.xlsxfile containing raw reads and edgeR normalized counts.

organism: Macaca fascicularis

source_name: kidney cortex

growth_protocol: Kidney cortex tissue obtained from naïve M. fascicularis (ages 2–3 years old)

extract_protocol:
Frozen kidney cortex samples (-80°C) were thawed on ice and used for total RNA extraction. Briefly, 25–30 mg of kidney cortex tissue were suspended in 700 μl QIAzol Lysis Reagent (Qiagen) and homogenized using TissueLyser for 90 seconds. Total RNA was extracted from the tissue homogenate using miRNeasy mini kit (Qiagen).
Five micrograms of total RNA were used for small RNA enrichment. Small RNA enrichment  was performed using Total RNA-Seq kit v2 (Life Technologies) as per manufacturer’s recommendations.  The sequencing library was prepared from 12 ng (quantified using High Sensitivity  DNA analysis kit and 2100 Bioanalyzer) of enriched small RNAs using Total RNA-Seq kit  v2 with custom barcode primers

value: kidney cortex

tag: stain

value: naïve


title: NAI (ESCs, Fibrillarin sh #1)

data_processing:
Library strategy: SHAPE-seq
Raw reads were converted to FastQ, and examined using the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For each run, the first 5 nucleotides at 5’-end were trimmed using the fastx_trimmer tool of the FASTX-Toolkit suite. Reads were then clipped from 3’ adapter sequences (requiring a minimum match of 10 nucleotides), using the fastx_clipper tool of the FASTX-Toolkit suite, discarding reads shorter than 25 nucleotides  (parameters: -a TGGAATTCTCGGGTGCCAAGG -l 25 -M 10 -Q 33; -a AGATCGGAAGAGCACACGTCT was replaced for Alkaline hydrolysis and 2OMe-seq Method #2 libraries, due to the different 3’ adapter used). Reads were mapped to the reference transcriptome, composed of 28S and 18S rRNA sequences, plus sequences of the 3 in vitro generated spike-ins (where included), using Bowtie v1.1.1 (parameters: -q -n 2 --norc -m 1 --best --strata).
Genome_build: Reads were mapped only to 18S and 28S rRNA sequence for each species.  Accessions are as follows: Homo sapiens 18S (NR_003286), Homo sapiens 28S (NR_003287), Mus musculus 18S (NR_003278), Mus musculus 28S (NR_003279).
Supplementary_files_format_and_content: Processed data file in BEDGraph format are provided (Columns: Transcript ID, Position, Position +1, Number of reads mapping 1nt downstream).

source_name: E14 Mouse Embryonic Stem Cells

growth_protocol: Mouse embryonic stem cells E14 were grown on 0.1% gelatin-coated plates and maintained in DMEM (4.5g/L D-Glucose), supplemented with 15% heat-inactivated FBS, 0.1mM NEAA, 1mM Sodium Pyruvate, 0.1mM 2-Mercaptoethanol, 25U/ml penicillin, 25 μg/ml streptomycin and 1,500U/ml LIF, as previously described (Neri et al., 2013).

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
Libraries for NAI-treated RNA were prepared with minor changes to the protocol used for CIRS-seq (Incarnato et al., 2014). Briefly, 2 μg of total RNA were denatured at 70°C for 5 minutes, then stored immediately on ice. RNA was treated with 100mM NAI for 20 minutes at 37°C with moderate shaking. Reaction was quenched by addition of 0.7M 2-Mercaptoethanol and purified on RNeasy Micro Spin columns (Qiagen). Reverse transcription was performed using 200U of SuperScript III Reverse Transcriptase, and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). The reverse transcription reaction was conducted in 50 minutes at 50°C, followed by 15 minutes at 75°C to inactivate the reverse transcriptase. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs recovery, and excess adapter removal was performed using Agencourt Ampure XP beads (Beckman Coulter). Following cleanup, cDNAs were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 50-200nt fragments was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. The adapter-ligated cDNAs were then subjected to 15 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.

treatment_protocol: Custom shRNAs were constructed using the TRC hairpin design tool (http://www.broadinstitute.org/rnai/public/seq/search), and designed to target the 3’-UTR of the Fbl transcript (NM_007991). shRNAs with more than 14 consecutive matches to non-target transcripts were avoided. Hairpins were cloned into pLKO.1 vector (Addgene: 10878) and each construct was verified by sequencing. For mouse ESCs transfection, approximately 106 cells were seeded in a 6-well plate the day prior to transfection. Transfection was performed using Lipofectamine 2000 reagent (Invitrogen), following manufacturer protocol. Cells were maintained for 4 days in canonical medium supplemented with Puromycin (2 μg/ml for 1 day, 1 μg/ml for 3 days).

value: E14 Mouse Embryonic Stem Cells


title: BC19 K3


title: NAI (ESCs, Fibrillarin sh #2)


title: BC18 K2


title: NAI (ESCs, Ctrl)


title: 2OMe-seq 1mM dNTPs (ESCs, Fibrillarin sh #2)

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
For each experiment, 2 μg of total RNA were spiked-in with 100 ng of in vitro transcribed RNAs (where specified). 2OMe-seq libraries were prepared with minor changes to the protocol used for CIRS-seq (Incarnato et al., 2014). Briefly, reverse transcription was performed in a 20 μl reaction, using 1mM final dNTPs, 0.5U/μl AMV Reverse Transcriptase (NEB), and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs recovery, and excess adapter removal was performed using Agencourt Ampure XP beads (Beckman Coulter). Following cleanup, cDNAs were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 70-300nt fragments was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. This approach allowed us to keep the strand-specificity of the library, so that each read started 1 nt downstream of the RT stopping point. The adapter-ligated cDNAs were then subjected to 15 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.


title: 2OMe-seq 0.004mM dNTPs (ESCs, Fibrillarin sh #1)

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
For each experiment, 2 μg of total RNA were spiked-in with 100 ng of in vitro transcribed RNAs (where specified). 2OMe-seq libraries were prepared with minor changes to the protocol used for CIRS-seq (Incarnato et al., 2014). Briefly, reverse transcription was performed in a 20 μl reaction, using 0.004mM final dNTPs (Low dNTP sample), 0.5U/μl AMV Reverse Transcriptase (NEB), and random hexamers fused with the reverse complement of the Illumina TruSeq RNA 3’ Adapter (CCTTGGCACCCGAGAATTCCANNNNNN). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs recovery, and excess adapter removal was performed using Agencourt Ampure XP beads (Beckman Coulter). Following cleanup, cDNAs were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 70-300nt fragments was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. This approach allowed us to keep the strand-specificity of the library, so that each read started 1 nt downstream of the RT stopping point. The adapter-ligated cDNAs were then subjected to 15 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.


title: 2OMe-seq 0.004mM dNTPs (ESCs, Ctrl)


title: 2OMe-seq 1mM dNTPs (ESCs, Ctrl)


title: NCCIT_Input

city: BALTIMORE

first: hari

last: easwaran

state: MD

address: 1650 Orleans Street, CRB1, Room 530

department: Oncology

institute: Johns Hopkins University

description: No immunoprecipitation control

data_processing:
Reads were aligned to hg18 (NCBI36) using Bioscope 1.2.1, finding all alignments between the first 25 bp of the read (seed) and the reference sequence with up to two mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and −2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported.
Samtools and Bedtools used to filterreads as below and get output as BED files: Remove duplicates and create bam file (-b) of only mapped reads (-F 4) with mapping quality (MAPQ) better than 20 (-q 20), and not more than 1 mismatches (NM is 0 or 1; NM is the number of bases that has to be edited to get a perfect match).
Peaks were called using SICER. Settings for finding H3K4me3 peaks: redundancy_threshold=1, window_size_bp=200, fragment_size=160, effective_genome_fraction=0.75, gap_size_bp=600, FDR=0.01;  Settings for H3K27me3 peaks:redundancy_threshold=1, window_size_bp=200, fragment_size=160, effective_genome_fraction=0.75, gap_size_bp=1000, FDR=0.01
Genome_build: hg18
Supplementary_files_format_and_content: *.txt file; summary of all candidate islands with their statistical significance. File format/column labels: chrom, start, end, ChIP_island_read_count, CONTROL_island_read_count, p_value, fold_change, FDR_threshold

source_name: NCCIT cells

growth_protocol: NCCIT cells were grown in RPMI (Corning) with 15% FBS. All cells were grown in 5% CO2 at 37°C to 70-80% confluence in 150 mm culture dishes.

extract_protocol:
Mononucleosomes were isolated by MNase digestion of cells, followed by formaldehyde fixing and fractionating the concentrated nucleosome mixture it on a 5-25% sucrose gradient, to purify the mononucleosome fraction away from dinucleosomes. Alternate "Quick protocol" involved controlled detergent mediated treatment of NCCIT cells and digestion with a fixed concentration of MNase that allows release of mononucleosomes into the supernatant that are crosslinked and used for ChIP. Modified Sequential ChIP involved crosslinking of the 1st Antibody to beads by DSG followed by elution with SDS/65degC and the elute used for sequential pulldown using the 2nd Antibody. DNA was extracted using Qiagen PCR purification preps from the decrosslinked eluate of sequential ChIP and was used either for western blotting or PCR.
10ng of seq ChIP DNA was amplified by WGA using Illumina kits for Library preparation. Libraries were constructed according to the protocol provided by Illumina’s TruSeq DNA Sample Preparation v2 Kit.  Library prep begins with the End Repair and dA tailing followed by Illumina indexed sequencing adaptor ligation that are complementary for hybridization on the flow cell.  PCR amplification was used to amplify the DNA fragments for 15 cycles and assessment of the yield and size distribution of the amplified library was performed on the Agilent 2100 Bioanalyzer using the High Sensitivity Chip.  Quantitation of the libraries was performed by qPCR using the KAPA SYBER FAST qPCR Kit supplied by Kapa Biosystems according to manufactures’ protocol.  Multiplex libraries were pooled to a final concentration of 2nM and cluster generation completed on Illumina’s cBot using the TruSeq SR Cluster Kit v3.  Single read 50bp sequencing was performed on the Illumina HiSeq2000 with the TruSeq SBS Kit v3 (50cycles).

treatment_protocol: No treatment

value: NCCIT

value: Pluripotent embryonal carcinoma

tag: antibody

value: No antibody


title: E11.5 Control 92-1

city: Taipei

first: Pao-Yang

last: Chen

address: 128 Sec. 2, Academia Rd, Nankang,

department: Institute of Plant and Microbial Biology

country: Taiwan

institute: Academia Sinica

description:
E11.5 female WT rep4
RPKM_table_E11_5.txt

data_processing:
RNA-seq alignment using Tophat 2.1.0 with default parameters
Calculate RPKM and by using cuffdiff (cufflinks-2.2.1) with default parameters
Genome_build: mm9
Supplementary_files_format_and_content: output from cuffdiff, RPKM values for WT and MT

growth_protocol: Embryos were obtained from crosses between Oct4-IRES-Gfp (OG) (Lengner et al., 2007); Dnmt12lox (Jackson-Grusby et al., 2001) homozygous females and Blimp1-Cre (BC) males.  Embryos were staged by the detection of a vaginal plug on the morning after time-mating pairs were established (0.5 dpc).

extract_protocol:
GFP positive PGCs were sorted and collected in a 1.5ml microcentrifuge tube containing 350ul of RLT buffer.  RNA was extracted using the RNeasy Micro Kit (Qiagen).
The RNA was subsequently amplified through the use of the Ovation RNA-Seq System V2 kit (Nugen), and bar codes were added using the Encore Rapid Library Systems kit (Nugen).

tag: developmental stage

value: primordial germ cells

tag: genotype

value: wild type

value: female


title: E11.5 Control 89-4

description:
E11.5 male WT rep2
RPKM_table_E11_5.txt

value: male


title: E11.5 DCKO 92-2

description:
E11.5 male Dnmt1 KO rep4
RPKM_table_E11_5.txt

value: Dnmt1 -/-


title: E11.5 Control 85-4

description:
E11.5 female WT rep1
RPKM_table_E11_5.txt


title: E11.5 DCKO 89-2

description:
E11.5 male Dnmt1 KO rep3
RPKM_table_E11_5.txt


title: 2OMe-seq 0.004mM dNTPs (ESCs, Fibrillarin sh #2)


title: E11.5 DCKO 89-1

description:
E11.5 male Dnmt1 KO rep2
RPKM_table_E11_5.txt


title: E11.5 Control 92-3

description:
E11.5 male WT rep4
RPKM_table_E11_5.txt


title: E11.5 DCKO 91-2

description:
E11.5 female Dnmt1 KO rep5
RPKM_table_E11_5.txt


title: NCCIT_Sequential ChIP-seq

description: Sequential ChIP-seq with anti-H3K27me3 followed by anti-H3K4me3

value: anti-H3K4me3 (07-473, Millipore) and  anti-H3K27me3 (07-449, Millipore)


title: E11.5 DCKO 90-3

description:
E11.5 female Dnmt1 KO rep4
RPKM_table_E11_5.txt


title: NCCIT_H2AZ_ChIP-Seq

description: ChIP-seq with mentioned Ab

value: anti-H2A.Z (ab4174, Abcam)


title: E11.5 Control 87-1

description:
E11.5 female WT rep2
RPKM_table_E11_5.txt


title: E11.5 Control 90-4

description:
E11.5 female WT rep3
RPKM_table_E11_5.txt


title: E11.5 Control 89-3

description:
E11.5 male WT rep1
RPKM_table_E11_5.txt


title: EGS_sup-2

city: Berkeley

first: Xavier

last: Darzacq

state: CA

address: 475D Li Ka Shing Center

department: Molecular and Cell Biology

institute: University of California, Berkeley

data_processing:
Library strategy: Micro-C XL
OLB 1.9/CASAVA_v1.8.2 were used for base calling
Barcoded libraries were demultiplexed by Novobarcode.
Paired reads were mapped to reference genome separately by Bowtie2
Parse SAM files and remove duplicate reads.
Mate unique paired mates and generate contact matrix.
Genome_build: S288C (sacCer3) or S. pombe 972h- (ASM294v2)
Supplementary_files_format_and_content: Interaction_density.xlsx

organism: Saccharomyces cerevisiae

source_name: Yeast

growth_protocol: The 100mL yeast cultures were grown to midlog phase ~OD 0.55 in indicated media at 30 C.

extract_protocol:
DNA was purified by PCI extraction and ethanol precipitation, treated with RNase A, and ~250-350 bp DNA was gel-purified following 3% agarose gel electrophoresis.
Purified DNA was treated with End-it, subject to A-tailing with Exo- Klenow, and ligated to Illumina adaptors. Adaptor-ligated DNA was then purified with streptavidin beads to isolate ligated Micro-C products away from undigested dinucleosomal DNA. Streptavidin beads were then subject to ~10-13 cycles of PCR using Illumina paired-end primers.
Amplified library was purified and subject to Illumina Nextseq 500 paired end sequencing with 40 x 40 bp reads.

treatment_protocol: 1) Protocol for formaldehyde only: Yeast cells were fixed with 3% formaldehyde for 15 min and quenched with 125 mM glycine for 5 min. Cells were pelleted, spheroplasted with Zymolyase, and treated with a level of MNase yielding >95% mononucleosomes. 2) Protocol for long crosslinking: Yeast cells were fixed with 3% formaldehyde for 10 min, and quenched with 125 mM glycine for 5 min. Cells were pelleted and spheroplasted with Zymolyase. The spheroplasts were washed by PBS, subjected to long-crosslinker fixation for 40min, and quenched with 250 mM glycine for 5 min. Cells were pelleted again, washed with PBS, and treated with a level of MNase yielding >95% mononucleosomes. After stopping MNase, chromatin pellet and supernatant was dephosphorylated using rSAP separately. Crosslinked chromatin was subject to T4 DNA polymerase / T4 PNK enzyme mix solution to generate blunt ends. Chromatin was diluted into 2.5 mL and treated with T4 DNA ligase. Ligated products were treated with 100U of exonuclease III for 5 min to eliminate biotinylated ends of unligated DNA. Proteinase K was then added and incubated at 65 C overnight.

tag: media

value: YPD

tag: first fixation

value: 3% Formaldehyde for 10 min

tag: second fixation

value: 3mM EGS for 40min


title: EGS_sup-1


title: E11.5 DCKO 88-2

description:
E11.5 male Dnmt1 KO rep1
RPKM_table_E11_5.txt

tag: gender


title: 2OMe-seq 1mM dNTPs (ESCs, Fibrillarin sh #1)


title: E11.5 Control 90-2

description:
E11.5 male WT rep3
RPKM_table_E11_5.txt


title: EGS_pellet-2


title: E11.5 DCKO 87-3

description:
E11.5 female Dnmt1 KO rep3
RPKM_table_E11_5.txt


title: E11.5 DCKO 87-2

description:
E11.5 female Dnmt1 KO rep2
RPKM_table_E11_5.txt


title: NCCIT_H3K4me3_ChIP-Seq

description: ChIP-seq with mentioned Ab

value: anti-H3K4me3 (07-473, Millipore)


title: GF_RNA-seq

city: Los Angeles

first: Masafumi

last: Horie

address: 2011 Zonal Ave

institute: University of Southern California

data_processing:
Raw sequence reads were mapped to the hg38 genome by Tophat (version 2.0.14)
Cufflinks  (version 2.0.2) were applied for estimation of FPKM.
Genome_build: hg38
Supplementary_files_format_and_content: .fpkm_tracking

source_name: gingival fibroblasts

extract_protocol:
not provided
rRNA depletion was performed with 1μg total RNA using TruSeq Stranded Total RNA with Ribo-Zero GoldKit (Illumina Inc. San Diego, USA). Prepared libraries were sequenced with 2 x 100 bp paired-end reads on the Illumina HiSeq 2500 sequencer (Illumina).

value: gingival fibroblasts


title: EGS_pellet-1


title: E11.5 DCKO 85-2

description:
E11.5 female Dnmt1 KO rep1
RPKM_table_E11_5.txt


title: NCCIT_H3K27me3_ChIP-Seq

description: ChIP-seq with mentioned Ab

value: anti-H3K27me3 (07-449, Millipore)


title: DSG_sup-3

value: 3mM DSG for 40min


title: E11.5 DCKO 92-5

description:
E11.5 female Dnmt1 KO rep6
RPKM_table_E11_5.txt


title: DSG_sup-2


title: DSG_sup-1


title: E10.5 Control 67-2

description:
E10.5 male WT rep2
RPKM_table_E10_5.txt


title: E10.5 Control 57-5

description:
E10.5 male WT rep1
RPKM_table_E10_5.txt


title: E10.5 Control 58-1

description:
E10.5 female WT rep1
RPKM_table_E10_5.txt


title: E10.5 Control 67-4

description:
E10.5 female WT rep2
RPKM_table_E10_5.txt


title: E10.5 DCKO 67-3

description:
E10.5 male Dnmt1 KO rep1
RPKM_table_E10_5.txt


title: FA3_sup-2

value: 3% Formaldehyde for 15 min


title: DSG_pellet-1


title: DSG_pellet-2


title: E10.5 DCKO 67-1

description:
E10.5 female Dnmt1 KO rep2
RPKM_table_E10_5.txt


title: DSG_pellet-3


title: E10.5 DCKO 58-3

description:
E10.5 female Dnmt1 KO rep1
RPKM_table_E10_5.txt


title: OE17

city: Rehovot

first: Tsviya

last: Olender

address: 234 Herzl St.

country: Israel

institute: The Weizmann Institute

description: human olfactory epithelium RNAseq

data_processing:
Basecalls performed using CASAVA version 1.8
Reads were aligned to the genome with TopHat 2.0.4
Genome_build: hg19
Supplementary_files_format_and_content: wig files were calculated with bam2wig.py script

source_name: olfactory epithelium, biopsy

extract_protocol:
Human olfactory epithelium biopsies were dissected from the superior ethmoturbinal, the cribriform plate and the superior septum.
Libraries were prepared using the Illumina mRNA-seq Sample Preparation Kit (San Diego, CA) and validated with an Agilent Bioanalyzer (Santa Clara, CA).

tag: Sex

value: olfactory epithelium


title: OE15

description: human olfactory epithelium RNAseq


title: FA3_pellet-1


title: E13.5 Male Control rep3

description:
male WT rep3
male_gene_exp.diff

data_processing:
RNA-seq alignment using Tophat 2.1.0 with default parameters
Calculate RPKM and by using cuffdiff (cufflinks-2.2.1) with default parameters
Genome_build: mm9
Supplementary_files_format_and_content: output from cuffdiff, RPKM values for WT and MT, p-value for DEG

extract_protocol:
GFP positive PGCs at E13.5 were sorted and collected in a 1.5ml microcentrifuge tube containing 350ul of RLT buffer.  RNA was extracted using the RNeasy Micro Kit (Qiagen).
The RNA was subsequently amplified through the use of the Ovation RNA-Seq System V2 kit (Nugen), and bar codes were added using the Encore Rapid Library Systems kit (Nugen).


title: FA3_pellet-2


title: OE12

description: human olfactory epithelium RNAseq


title: E13.5 Male Control rep1

description:
male WT rep1
male_gene_exp.diff


title: Input_DNA

city: La Jolla

first: Kyung Won

last: Kim

address: 9500 Gilman Drive

department: Section of Neurobiology, Division of Biological Sciences

institute: University of California, San Deigo

description: ChIP antibody: none

data_processing:
ChIP-seq reads were aligned to the ce10 genome assembly using CLC genomics workbench 6.0
Each read was Trimmed with the following settings: Removal of terminal nucleotides(nt): 1 nt from the 5' end and 5 nt from the 3' end; Removal of ambiqious nt: maximal 2 nt allowed; removal of low quality sequence (limit = 0.05).
Trimmed reads were mapped to the C. elegans genome reference (ce10).
Peaks were called using CLC genomics workbench 6.0 ChIP-seq Analysis tool.
Genome_build: ce10
Supplementary_files_format_and_content: tab-delimited text file was generated using CLC genomics workbench 6.0; file includes peak location and annotation of nearby gene within 3 kb.

organism: Caenorhabditis elegans

source_name: C. elegans whole worm lysate

growth_protocol: Transgenic C. elegans animals expressing FLAG-tagged CEBP-1 in a cebp-1(tm2807) mutant background (cebp-1(0); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3’UTR]) were grown at 20˚C on NGM plates.

extract_protocol:
FLAG::CEBP-1-DNA complexes were isolated with anti-FLAG antibody.
Both ChIPed DNA and input genomic DNA were ligated to specific adaptors and amplified by barcode primers. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo minus) and dATP to yield a protruding 3- 'A' base for ligation of adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with primers for 12 (1st) or 18 (2nd) cycles with DNA polymerase (KAPA) and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was quantified using Qubit. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

treatment_protocol: Mixed stage worms were collected followed by 2% formaldehyde and sonicated.

value: mixed stage

value: cebp-1(tm2807); juIs418 [Pcebp-1::FLAG::CEBP-1::cebp-1 3'UTR]


title: CEBP-1_ChIP

description: ChIP antibody: FLAG (M2 anti-FLAG magnetic beads; Sigma, M8823)


title: E13.5 Male Control rep2

description:
male WT rep2
male_gene_exp.diff

value: primordial germ cells

value: wild type


title: FA3+EGS


title: FA3+DSG


title: FA3_ctrl


title: E13.5 Male DCKO rep1

description:
male Dnmt1 KO rep1
male_gene_exp.diff

value: male


title: Input_dTSCs

city: Imabari-shi

first: Koji

last: Hayakawa

state: Ehime

address: 1-3 Ikoino-oka

department: Department of Toxicology, Faculty of Veterinary Medicine

country: Japan

institute: Okayama University of Science

data_processing:
In this study, all of data processing was performed by using Galaxy platform (https://usegalaxy.org/).
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie (version 1.1.2).
Peaks were called using MACS (ver 1.0.1) with the following setting: Tag size (100), Band width (125), Pvalue cutoff for peak detection (1e-5).
Genome_build: mm9
Supplementary_files_format_and_content: wig files and bed files were generated using MACS (ver1.0.1) with the following setting: Extend tag from its middle point to a wigextend size fragment (-1), Resolution for saving wiggle files (10).

source_name: Differentiated trophoblast stem cells

growth_protocol: TSCs maintained stemness in RPMI-1640 supplemented 20% fetal bovine serum, 2 mM L-glutamine, 1 mM Na-pyruvate, 25 ng/mL FGF4, 1 μg/mL Heparine and 10 ng/mL Activin. Differentiation of TSCs was induced by cultured without FGF4, heparine and Activin for 6 days.

extract_protocol:
ChIP-enriched DNA was obtaind by using ChIP-IT Express Enzymatic (Active Motif, Cat No. 53009) and S40Gc specific antibody (20B2)
The ends of 50 ng of ChIP-enriched DNA and Input DNA were repaired using T4 DNA polymerase (New England Biolabs) and phosphorylated with T4 polynucleotide kinase (New England Biolabs). A single adenine base was added to the 3 prime-end with Klenow fragments (New England Biolabs). TruSeq DNA adapters (Illumina) were ligated to the fragments with T4 DNA ligase (New England Biolabs). Ligation products between 200 and 600 bp were purified on AMpure XP beads (Beckman Coulter Inc.) to remove unligated adapters and subjected to 14 cycles of PCR amplification by KAPA Library Amplification Kit (KAPA BIOSYSTEMS). Completed libraries were quantified using an Agilent 2000 BioAnalyzer system.

value: TSC

tag: cell differentiation state

value: Differentiated

tag: chip-antibody


title: E13.5 Male DCKO rep3

description:
male Dnmt1 KO rep3
male_gene_exp.diff

value: Dnmt1 -/-


title: FA1+DSG+EGS

value: YPD

value: 1% Formaldehyde for 10 min

value: 3mM DSG and EGS for 40min


title: FA1+EGS

value: 3mM EGS for 40min


title: E13.5 Male DCKO rep2

description:
male Dnmt1 KO rep2
male_gene_exp.diff


title: FA1+DSG


title: E13.5 Female Control rep2

description:
female WT rep2
female_gene_exp.diff

value: female


title: Clindamycin_replicate_3

city: Chicago

first: Mee-Ngan

middle: F.

last: Yap

state: IL

address: 320 E Superior St

department: Microbiology-Immunology

institute: Northwestern University Feinberg School of Medicine

data_processing:
Basecalling was performed by Torrent Suite version 5 software using the default settings.
Alignment to the E. coli K12 MG1655 genome sequence was performed using TMAP map4 algorithm with 5' and 3' soft-clipping and a minimum seed length of 20 nt
The total sequencing base pair coverage for all annotated genes was summed for each sample and normalized to total coverage using bedtools and custom R scripts as described in Wu et al. 2015 PLoS Genetics Nov 6;11(11):e1005655.
Genome_build: Escherichia coli str. K-12 substr. MG1655, NCBI Reference Sequence: NC_000913.3
Supplementary_files_format_and_content: Microsoft Excel File with normalized sequencing coverage of all annotated genes and statistical comparison of expression in antibiotic treated to untreated cells .

organism: Escherichia coli

source_name: E. coli strain K12

growth_protocol: E. coli strain NM580 (genotype MG1655 ermBL-ermB’::LacZ) cells were grown in LB broth (37°C) until OD600 of ~0.3.

extract_protocol:
Briefly, hot phenol-chloroform extraction was done by mixing the cell culture in ½ volume of 99°C lysis solution (2% SDS, 16 mM EDTA, 200 mM NaCl made in RNAse-free H2O) for 10 min. The suspension was extracted twice with 1 volume of 65°C acid phenol/chloroform (pH 4.5), once in chloroform/isoamyl alcohol, and precipitated with isopropanol. Pellets were washed with 1 mL of 70% ethanol, dried and purified using QIAGEN RNeasy kit. Final RNA was eluted with 100 µL of nuclease-free water. Following purification, DNAse treatment with TURBO DNase kit was performed according to manufacturer’s manual, except that twice amount of recommended DNase inactivation reagent was used. The integrity of DNA-free RNA was analyzed on either native agarose gel or on Agilent Bioanalyzer RNA 6000 Nano chip.
Ion Xpress barcoded libraries were constructed using the Ribo-Zero Magnatic Kit (Gram-negative bacteria, Epicentre )and IonXpress RNA-seq v2 (Life Technologies) kits according to the manufacturer's directions.

treatment_protocol: The cells were untreated, or treated with  (100µg/mL Erythromycin or 50 µg/mL Clindamycin in 70% Ethanol). After 10 minutes at 37°C, 1 mL samples were subjected to hot phenol-chloroform extraction. Ref: Chuang SE, Daniels DL, and Blattner FR (1993) Global regulation of gene expression in Escherichia coli. J. Bacteriol. 175:2026-2036.

tag: treatment

value: clindamycin


title: S40Gc_dTSCs_ChIPseq

tag: strain

value: S40Gc specific antibody 20B2 (mouse monoclonal antibody, made in-house)


title: OE7

description: human olfactory epithelium RNAseq


title: S40Gc_TSCs_ChIPseq

source_name: Trophoblast stem cells

value: Undifferentiated


title: EGS_sup-1 [S. pombe]

organism: Schizosaccharomyces pombe

value: Compromised Media


title: E13.5 Female DCKO rep3

description:
female Dnmt1 KO rep3
female_gene_exp.diff


title: Clindamycin_replicate_1


title: E13.5 Female Control rep3

description:
female WT rep3
female_gene_exp.diff


title: Clindamycin_replicate_2


title: FA3+DSG+EGS

value: 3% Formaldehyde for 10 min


title: FA1_ctrl

value: 1% Formaldehyde for 15 min


title: EGS_pellet-1 [S. pombe]


title: E13.5 Female Control rep1

description:
female WT rep1
female_gene_exp.diff

value: primordial germ cells


title: E13.5 Female DCKO rep1

description:
female Dnmt1 KO rep1
female_gene_exp.diff


title: E13.5 Female DCKO rep2

description:
female Dnmt1 KO rep2
female_gene_exp.diff


title: Erythromycin_replicate_3

value: erythromycin


title: Input_TSCs


title: Erythromycin_replicate_2


title: DSG_pellet-2 [S. pombe]

value: 3mM DSG for 40min


title: FA3_sup-1

value: 3% Formaldehyde for 15 min


title: Alkaline hydrolysis (HeLa)

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
Briefly, 2 μg of total RNA were resuspended in 50mM Na2CO3 buffer, and heated 6 minutes at 95°C. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was dephosphorylated by treating with 5U Antarctic Phosphatase (NEB) at 37°C for 30 minutes, followed by 10 minutes at 65°C to inactivate the enzyme. Dephosphorilated RNA fragments were end repaired by adding 20U of T4 PNK (NEB), and incubating at 37°C for 1h, and then purified again on RNA Clean & Concentrator™-5 columns. A pre-adenylated (rApp) adapter (AGATCGGAAGAGCACACGTCT) was ligated to the end-repaired RNA fragments using 400U of T4 RNA Ligase 2, Deletion Mutant (Epicentre) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The day after, RNA fragments were purified on RNeasy Micro Spin columns (Qiagen) to remove the excess adapter, eluted, and then ligated to the Illumina TruSeq RNA 5’ Adapter (GUUCAGAGUUCUACAGUCCGACGAUC) using 20U of T4 RNA Ligase 1 (NEB), by incubating at 25°C for 2 hours. The adapter-ligated RNA was purified again on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter, and reverse transcription was performed in a 20 μl reaction, using 200U SuperScript III Reverse Transcriptase (Invitrogen), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). The reverse transcription reaction was conducted in 50 minutes at 50°C, followed by 15 minutes at 75°C to inactivate the reverse transcriptase. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNA fragments were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 80-200nt fragments was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, the adapter-ligated cDNAs were subjected to 15 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.

tag: cell line

value: HeLa S3

value: cervical adenocarcinoma


title: DSG_pellet-1 [S. pombe]


title: Erythromycin_replicate_1


title: Untreated_replicate_3

value: untreated


title: 2OMe-seq 0.004mM dNTPs (HeLa, Method #2)

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
Briefly, 2 μg of total RNA were fragmented by addition of 5X First Strand Buffer (Invitrogen), and incubation at 95°C for 5 minutes. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was end repaired by adding 20U of T4 PNK (NEB), and incubating at 37°C for 1h, and then purified again on RNA Clean & Concentrator™-5 columns. A pre-adenylated (rApp) adapter (AGATCGGAAGAGCACACGTCT) was ligated to the end-repaired RNA fragments using 400U of T4 RNA Ligase 2, Deletion Mutant (Epicentre) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The day after, reactions were purified on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter. Following cleanup, adapter-ligated RNA fragments were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 200-250nt fragments was cut. RNA was recovered by passive diffusion in RNA Diffusion Buffer [450mM Ammonium Acetate, 0.05% SDS] for 16 hours at 4°C with end-to-end rotation. Following isopropanol precipitation, reverse transcription was performed in a 20 μl reaction, using 0.004mM final dNTPs (Low dNTP sample), 0.5U/μl AMV Reverse Transcriptase (NEB), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs were purified on RNA Clean & Concentrator™-5 columns (Zymo Research), separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 50-150nt fragments (corresponding to truncated reverse transcription products) was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. The adapter-ligated cDNAs were then subjected to 18 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.


title: 2OMe-seq 1mM dNTPs (HeLa, Method #2)

extract_protocol:
Cells were washed twice in 1X PBS, and lysed by addition of 1ml ice-cold TRIzol (Invitrogen). RNA integrity measurements were performed using Fragment Analyzer™ (Advanced Analytical). All samples had RNA Quality Number (RQN) greater than 9.8.
Briefly, 2 μg of total RNA were fragmented by addition of 5X First Strand Buffer (Invitrogen), and incubation at 95°C for 5 minutes. Fragmented RNA was purified on RNA Clean & Concentrator™-5 columns (Zymo Research). The eluted RNA was end repaired by adding 20U of T4 PNK (NEB), and incubating at 37°C for 1h, and then purified again on RNA Clean & Concentrator™-5 columns. A pre-adenylated (rApp) adapter (AGATCGGAAGAGCACACGTCT) was ligated to the end-repaired RNA fragments using 400U of T4 RNA Ligase 2, Deletion Mutant (Epicentre) in the presence of 20% PEG-8000, by incubating at 16°C for 18 hours. The day after, reactions were purified on RNeasy Micro Spin columns (Qiagen) to get rid of the excess adapter. Following cleanup, adapter-ligated RNA fragments were separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 200-250nt fragments was cut. RNA was recovered by passive diffusion in RNA Diffusion Buffer [450mM Ammonium Acetate, 0.05% SDS] for 16 hours at 4°C with end-to-end rotation. Following isopropanol precipitation, reverse transcription was performed in a 20 μl reaction, using 1mM final dNTPs (High dNTP sample), 0.5U/μl AMV Reverse Transcriptase (NEB), and an oligonucleotide complementary to the 3’ adapter (AGACGTGTGCTCTTCCGATCT). The reverse transcription reaction was conducted in 30 minutes at 42°C, followed by 10 minutes at 95°C to inactivate the AMV enzyme. Template RNA was degraded by addition of 1 μl of Ribonuclease H (Ambion), and 1 μl of RNase Cocktail Enzyme Mix (Ambion), followed by 30 minutes incubation at 37°C. cDNAs were purified on RNA Clean & Concentrator™-5 columns (Zymo Research), separated on a 10% TBE-Urea PAGE gel, and a gel slice corresponding to 50-150nt fragments (corresponding to truncated reverse transcription products) was cut. DNA was recovered by passive diffusion in TE Buffer [10mM Tris-HCl, 1mM EDTA] for 16 hours at 37°C with moderate shaking. Following ethanol precipitation, an adapter corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5’ Adapter (GATCGTCGGACTGTAGAACTCTGAAC), modified with a 5’-P group and a 3’-C3 spacer, was ligated to cDNAs 3’-OH termini using 200 U of CircLigase II for 6 hours at 65°C. The adapter-ligated cDNAs were then subjected to 18 cycles of PCR using standard Illumina TruSeq primers, and excess primers were removed using Agencourt Ampure XP beads. Libraries were pooled in equimolar amounts, and subjected to sequencing on the Illumina™ NextSeq 500 Sequencer.


title: Untreated_replicate_2


title: 2OMe-seq 0.004mM dNTPs (HeLa #1, Method #1)


title: Untreated_replicate_1


title: 2OMe-seq 1mM dNTPs (HeLa #1, Method #1)