title: ESC +pRNA -H3K9me2 repl2

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

department: Dep. of Molecular Mechanisms  of Disease

country: Switzerland

institute: University of Zurich

data_processing:
Fastaq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
The bam files were analyzed using the damidseq pipeline script from the Brand group (http://owenjm.github.io/damidseq_pipeline) (Marshall and Brand, 2015). The pipeline bins the mapped reads into GATC-fragments according to GATC-sites indicated by a gff file for the GRCm38 mouse genome (already provided by the authors of the pipeline on the website above) and normalizes reads against the Dam control, in our case the H2B-Dam sample. The pipeline gives a bedgraph file with the log2 ratio of the m6A between H2B-Dam-NoLS and the H2B-Dam only.  The bedgraph files were processed with the find_peaks software associated with the pipeline (https://github.com/owenjm/find_peaks) adjusting the values of the FDR and the minimum quantile (FDR <0.01 and min_quant 0.70). Only the significative peaks common to both replicates were considered as NADs for further analysis. The intersection of identified NADs and previously published lamina associated domains (LADs, Peric-Hupkes et al., 2010) results in the classification of NADonly, NAD/LAD, and LAD.
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
for RNA SEQ, reads were aligned to the reference genome (ensembl version 82) with Subread (i.e. subjunc, version 1.4.6-p4; 50) allowing up to 16 alignments per read (options: –trim5 10 –trim3 15 -n 20 -m 5 -B 16 -H –allJunctions). Count tables were generated with Rcount 51 with an allocation distance of 100 bp for calculating the weights of the reads with multiple alignments, considering the strand information, and a minimal number of 5 hits. For each gene, exon coverage was calculated using a custom pipeline and then normalized in reads per kilobase per million (RPKM) (Mortazavi et al., 2008), the method of quantifying gene expression from RNA sequencing data by normalizing for total read length and the number of sequencing reads.
Genome_build: mm10

source_name: Embryonic stem cells

growth_protocol: E14 mouse embryonic stem cells were cultured in either 2i media composed of DMEM-F12 and Neurobasal medium (1:1, Life Technologies), supplemented with 1× N2/B27 (Life Technologies), 1× penicillin/streptomycin/l-glutamine (Life Technologies), 50 μM β-mercaptoethanol (Life Technologies), recombinant leukemia inhibitory factor, LIF (Polygene, 1,000 U/ml) and MEK and GSK3β inhibitors, 2i (Sigma CHIR99021 and PD0325901, 3 and 1 μM, respectively). ESCs were seeded at a density of 50,000 cells/cm2 in culture dishes (Corning® CellBIND® surface) coated with 0.1% gelatin without feeder layer. Propagation of cells was carried out every 2 days using enzymatic cell dissociation.

extract_protocol:
1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed with lysis buffer (50mM Tris-HCl, pH 8.1, 10 mM EDTA, pH 8, 1% SDS, 1X protease inhibitor cOmplete EDTA-free cocktail, Roche). Nuclei were sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and incubated overnight with the ChIP-grade antibodies against H3K9me2 and H3K9me3. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR.
The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) was used in the following steps. ChIP samples (1 ng) was end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).

treatment_protocol: 9 x 105 ESC were plated in gelatin-coated 10cm culture dish and transfected with 42 μg pRNA, or RNA-control using Lipofectamine MessengerMAX reagent (Invitrogen) in Opti-MEM GlutaMAX (Life Technologies) reduced-serum medium for 48h

tag: cell line

tag: antibody

tag: treatment

value: transfection with pRNA


title: m6A-seq FTO mutant shoot input rep2

status: Public on Aug 11 2021

type: SRA

city: Chicago

first: Shun

last: Liu

state: IL

address: 929 E. 57th. Street

department: Chemistry

country: USA

institute: University of Chicago

description:
FTOmut_shoot_rep2_gene_MeTPeak.txt
FTOmut_shoot_rep2_repeat_MeTPeak.txt

library_source: transcriptomic

data_processing:
Library strategy: m6A-seq
Adapters of raw reads were clipped by Cutadapt v1.15. Reads longer than 15 nt after trimming were mapped to the rice genome (MSU 7.0) using HISAT2 v2.1.0 with default parameters. mRNA and repeat annotation were downloaded from the Rice Genome Annotation Project and the Rice Annotation Project (RAP-DB), respectively.
For RNA-seq, reads on genes were counted by featureCounts (-t exon -g gene_id -C -M -O --fraction -T 24 -s 2 -p) from from Subread v1.6.4.
For m6A-seq, mapped reads were separated by strands and then MeTPeak was used to detect m6A peaks respectively with the mRNA and repeat anotation (FRAGMENT_LENGTH=200, READ_LENGTH=150, PEAK_CUTOFF_FDR=0.05, WINDOW_WIDTH=50, SLIDING_STEP=10, FOLD_ENRICHMENT=2).
Genome_build: MSU 7.0
Supplementary_files_format_and_content: Raw counts and peaks of mRNAs and repeats were present in tab-delimited plain text files

molecule: polyA RNA

organism: Oryza sativa

source_name: FTO transgenic mutant rice shoot

growth_protocol: Nipp and FTO rice were grown in Kimura B solutionKimura B solution  a 12-h light (26 °C)/12-h dark (22 °C) photoperiod, 50–60% relative humidity and 700 μmol m-2 s-1 light. 15 days old plants were harvested for experiment

extract_protocol:
For RNA-seq, 15-day-old seedlings of the Nipp and FTO plant were used for sequencing. Before purified non-ribosomal RNA with Ribo-Zero rRNA Removal Kit (Illumina), ERCC RNA spike-in control (Ambion) was added to each sample (0.1 μl per 5 ug total RNA). Library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, 1 g of 15-day-old seedlings of the Nipp, FTOmut and FTO plants from hydroponic experiments were used to isolate poly(A) RNA for m6A sequencing. 5 pg spike-ins for m6A seq mixture containing 3 diverse 20% m6A modified and 3 diverse non-m6A modified poly(A) spike-ins (see Supplementary Table 6) which synthesized in vitro used the MEGAscript ® Kit (AM1333, Thermo Fisher Scientific, USA) was added to the poly(A) RNA, followed by fragmented into ~100 nt using RNA Fragmentation Reagents (Ambion, USA) according to the manufacturer’s protocol.  m6A-IP was performed using EpiMark N6-Methyladenosine Enrichment Kit following the manufacturer’s protocol. Library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 4000 machine in pair-end mode with 150 bp per read (Genewiz, China).
For RNA-seq, library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7760S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China).

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

value: FTO homogeneous transgenic mutant

tag: ip antibody

tag: tissue

value: shoot


title: genomic DNA from ovarian cancer tissues [G685]

type: genomic

city: Pokfulam

first: David

middle: Wai

last: Chan

state: Hong Kong

address: L747 Laboratory Block, LKS Faculty of Medicine, 21 Sassoon Road

department: Obs. & Gyn.

country: Hong Kong

institute: The Uniersity of Hong Kong

description:
Ovarian tumor tissue
R03C01

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human MethylationEPIC Beadchip kit using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina iScan System

data_processing: Genome Studio Version 2011.1 and Methylation Fenome Studio Module v1.9

label: Cy5 and Cy3

molecule: genomic DNA

organism: Homo sapiens

source_name: Tumor Tissues

label_protocol: Standard Illumina Protocol

extract_protocol: genomic DNA was extracted and purified from LBC samples using Qiagen DNeasy Kit according to standard instructions

tag: Sex

value: female

tag: Stage

tag: grade

tag: age

tag: tumour subtypes

tag: tumour subtypes2

value: clear cell

tag: primary chemotherapy treatment

value: carboplatin/pacelitaxel

tag: recurrence

tag: doot

tag: dodx

tag: survivial status


title: ESC +RNA Ctrl -H3K9me2 repl2

library_source: genomic

organism: Mus musculus

tag: cell line

tag: antibody

value: transfection with RNA Ctrl


title: ESC +pRNA -H3K9me3 repl2

tag: treatment


title: genomic DNA from ovarian cancer tissues [G643]

description:
Ovarian tumor tissue
R07C01

tag: disease

value: ovarian cancer

value: serous

tag: platinum and drug treatment

tag: dls

tag: recurence date

tag: reccurrence within 5 years

value: Resistant


title: genomic DNA from ovarian cancer tissues [G644]

description:
Ovarian tumor tissue
R08C01

tag: Sex

tag: Stage

tag: grade


title: genomic DNA from ovarian cancer tissues [G638]

description:
Ovarian tumor tissue
R02C01

value: unstaged

value: unknown

tag: age

tag: tumour subtypes


title: RNA-seq FTO mutant root rep1

description:
non-ribosomal RNA
root_gene_featureCounts.txt
root_repeat_featureCounts.txt

data_processing:
Adapters of raw reads were clipped by Cutadapt v1.15. Reads longer than 15 nt after trimming were mapped to the rice genome (MSU 7.0) using HISAT2 v2.1.0 with default parameters. mRNA and repeat annotation were downloaded from the Rice Genome Annotation Project and the Rice Annotation Project (RAP-DB), respectively.
For RNA-seq, reads on genes were counted by featureCounts (-t exon -g gene_id -C -M -O --fraction -T 24 -s 2 -p) from from Subread v1.6.4.
For m6A-seq, mapped reads were separated by strands and then MeTPeak was used to detect m6A peaks respectively with the mRNA and repeat anotation (FRAGMENT_LENGTH=200, READ_LENGTH=150, PEAK_CUTOFF_FDR=0.05, WINDOW_WIDTH=50, SLIDING_STEP=10, FOLD_ENRICHMENT=2).
Genome_build: MSU 7.0
Supplementary_files_format_and_content: Raw counts and peaks of mRNAs and repeats were present in tab-delimited plain text files

source_name: FTO transgenic mutant rice root

tag: ecotype

tag: genotype

tag: ip antibody

tag: tissue

value: root


title: genomic DNA from ovarian cancer tissues [G669]

description:
Ovarian tumor tissue
R01C01

tag: tumour subtypes2

tag: primary chemotherapy treatment

tag: recurrence

tag: doot

tag: dodx

tag: survivial status


title: ESC input repl2

tag: cell line

tag: antibody

tag: treatment


title: Cerebellar tissue - 84Q_rep1 [18_1_4]

city: Changhua

first: Chin-San

address: 135 Nan-Hsiao Street

country: Taiwan

institute: Changhua Christian Hospital

data_processing:
Basecalls performed using bcl2fastq version 2.20.
Both adaptor clipping and sequence quality trimming were performed using Trimmomatic v0.36 with a sliding-window approach
RNAseq reads were aligned to the mm10 genome assembly using hisat2 version 2.2.0 with default setting
Genome_build: mm10
Supplementary_files_format_and_content: raw counts files were generated using featureCounts implemented in Rsubread v1.22.2

molecule: total RNA

source_name: Cerebellar tissue - 84Q

growth_protocol: Tissue was harvest from 18-month old mice.

extract_protocol:
Total RNA was extracted using Trizol® Reagent (Invitrogen, USA) according to the instruction manual. Purified RNA was quantified at OD260nm using a ND-1000 spectrophotometer (Nanodrop Technology,USA) and qualitated by using a Bioanalyzer 2100 (Agilent Technology, USA) with RNA 6000 LabChip kit (Agilent Technology, USA).
Samples with very high RNA integrity numbers were selected for library construction, and the libraries were constructed using Agilent's SureSelect Strand-Specific RNA Library Preparation Kit followed by AMPure XP beads (Beckman Coulter, USA) size selection. Libraries were sequenced as paired-end 150 bp reads on an Illumina HiSeq 2500 (Illumina, San Diego, USA) with a depth of ~20 million reads each. Sequencing data (FASTQ reads) were generated using Welgene Biotech's pipeline based on Illumina's base-calling program bcl2fastq v2.20.

treatment_protocol: The SCA3 15Q and 84Q transgenic mice were administered intraperitoneal injections of PBS(0.01 M) or IGF1(50 mg/kg) weekly from the postnatal age of 9 months to 18 months.

tag: strain background

tag: genotype/variation

value: untreated

tag: cell type

value: cerebellar tissue


title: Cerebellar tissue - 84Q_rep2 [19_2_4]


title: set2-2_NETseq

city: Boston

first: Mary

middle: T

last: Couvillion

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

institute: Harvard Medical School

description: nascent RNA isolated by IP against Rpb3-3x FLAG affinity label

data_processing:
Library strategy: NET-seq
For NET-seq, libraries were sequenced in 76 base pair reads for all NextSeq libraires. For reads shorter than 75 basepairs respectively the adapter sequenice (ATCTCGTATGCCGTCTTCTGCTTG) was trimmed from the fastq files and files were filtered using PrinSeq version prinseq-lite-0.20.2 (http://prinseq.sourceforge.net). The linker contained a molecular barcode with a random hexamer sequence. These random hexamers were removed using custom python scripts. Data were then aligned to the SacCer3 genome using the TopHat2 aligner v2.0.5 and reverse transcription mispriming events are identified and removed where molecular barcode sequences correspond exactly to the genomic sequence adjacent to the aligned read. Only the position corresponding to the 5’ end of the sequencing read (after removal of the barcode), which corresponds to the 3’ end of the nascent RNA fragment, is recorded with a custom python script using HTSeq package in python.
For ChIP-nexus, all reads were filtered for the presence of the fixed barcode (CTGA), beginning at position 6 of the read. Next, the adapter sequence was trimmed using cutadapt with the following parameters: cutadapt -f fastq -a AGATCGGAAGAGCACACGTCT -O 3 -m 22 --length-tag ‘length=’. Lastly, before alignment to the genome, the molecular barcode was removed using custom python scripts. As the ChIP-nexus samples were spiked with S. pombe, the reads were aligned to a combined genome containing both the S. cerevisiae and S. pombe genomes. Reads were aligned using Bowtie version 1.1.1 (Langmead et al., 2009) with the following parameters: -v 2 -M 1 --best -S. Plus and minus strand coverage were then combined and coverage files were then generated using BEDtools version 2.23.0 with the following parameters: genomeCoverageBed -d -5. Only the 5’ end of the read, which corresponds to the point of crosslinking is recorded. The S. cerevisiae and S. pombe data were then separated and S. cerevisiae coverage files were normalized by the total number of mapped S. pombe reads.
Genome_build: sacCer3
Supplementary_files_format_and_content: Bedgraph with 4 columns: Chromosome, Start, End, Score

organism: Saccharomyces cerevisiae

source_name: Yeast

growth_protocol: Yeast strains were grown in YPD at 30°C with shaking from an initial OD of 0.05 to mid-log phase with an OD of 0.6-0.8.

extract_protocol:
For NET-seq, frozen cells were cryogenically pulverized for lysis and nascent RNA was isolated via an immunoprecipitation at 4°C of Rpb3-3xFLAG and purified using the miRNeasy kit (Qiagen, 217004). For ChIP-nexus cells were crosslinked in 1% formaldehyde, lysed by bead beating, and sonicated. Chomatin was isolated via immunoprecipitation using antibodies: for H3 (Abcam, ab1791), H4 (Abcam, ab10158), H3K23Ac (Millipore, 07-355), and H4K12Ac (Millipore, 07-595), IPs, 12 µg of each antibody were added to 100 µL of Protein G Dynabeads (Thermo Fischer Scientific) previously washed 3 times with 1 mL of 0.5% BSA in PBS.
For NET-seq, a preadenylated RNA linker was ligated onto the 3’ end of the immunoprecipitated RNA. Fragmentation of the ligated samples by alkaline hydrolysis followed by a size selection after electrophoresis on a polyacrylamide gel allowed for the final DNA library to contain inserts of a narrow range. cDNA synthesis by reverse transcription (SuperScriptsIII (Invitrogen)) was performed with a long 5’ phosohorylated primer split by two 18 carbon spacers. Circularization of the RT product was performed with CircLigase (Epicentre) according to the manufacturer’s directions. The PCR was performed directly on the circularized product resulting in DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site positioned so that sequencing would start at the 3’ end. DNA was purified from a PCR reaction that had not reached saturation (~8-10 cycles) by band extraction from a polyacrylamide gel. DNA was sequenced on the Illumina NextSeq 500 according to the manufacturer’s instructions in 75 base pair reads. Preparation of ChIP-nexus libraries was done as described in (He et al., 2015) with the following modifications made to adapt the protocol to yeast: Frozen cell pellets were thawed on ice 1600µL of LB 140 (50 mM HEPES pH 7.4, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1X protease inhibitors (Roche, cOmplete EDTA free)) was added to the pellet. The samples were then split into two screw-cap tubes and acid-washed glass beads (Sigma) were added to the meniscus. Cells were lysed by bead beating for a total of 8 minutes, beating for 1 minute at 4˚C followed by resting for 2 minutes on ice. Chromatin was then removed from the tubes by using a 25G needle to pierce the bottom of the tube. The tubes were then placed inside a 15 mL conical and spun for 1 minute at 1000 xg at 4˚C. The samples were then transferred to a 1.5 mL eppendorf tube and spun at 14,000 rpm for 5 minutes at 4˚C. Pellets were next washed in 800 µL of LB 140 and collected again by centrifuging at 14,000 rpm for 5 minutes at 4˚C. 800 µL of LB 140 was added to the sample and the suspension was transferred to a 15 mL polystyrene tube. 600 µL of LB 140 was used to wash the empty eppendorf tube and then added to the 15 mL tube. Chromatin was then sonicated using Diagenode Bioruptor. Six cycles of sonication were done, each for 5 minutes and 30 seconds ON and 30 seconds OFF for a total sonication time of 15 minutes. After sonication, the chromatin was spun at 20,000 xg for 20 minutes at 4˚C. The supernatant was transferred to a new tube and the concentration of the chromatin was assayed by a Modified Lowry assay (Pierce). The size of the sonicated chromatin was confirmed on a 1.5% agarose gel to be between 100 - 400 bp. For each IP, 3 mg of chromatin extract was used, spiked with 5% (by protein concentration) of S. pombe chromatin. Samples were diluted up to 10 mL using WB 140 (50mM HEPES pH 7.4, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). For H3 (Abcam, ab1791), H4 (Abcam, ab10158), H3K23Ac (Millipore, 07-355), and H4K12Ac (Millipore, 07-595), IPs, 12 µg of each antibody were added to 100 µL of Protein G Dynabeads (Thermo Fischer Scientific) previously washed 3 times with 1 mL of 0.5% BSA in PBS. After the final was, the supernatant was removed and the chromatin was added to the beads in a 15 mL conical tube. Samples were incubated overnight at 4˚C with rotation. The remainder of the library construction was done exactly as described in (He et al., 2015). The only modifications were changes made to the four wash buffers to adapt them to yeast. Wash buffer A consisted of 10mM TE and 0.1% Triton X-100. Wash buffer B consisted of 50mM HEPES pH 7.4, 140mM NaCl, 1mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate. Wash buffer C is the same as wash buffer B, but with 250mM NaCl. Wash buffer D consisted of 10mM Tris pH 7.5, 250 mM lithium chloride, 10 mM EDTA, 0.5% NP40, 0.1% sodium deoxycholate.

tag: strain

value: RPB3::RPB3-3xFLAG NAT, SET2::HIS3

tag: rna fraction

value: Nascent RNA

tag: ip

value: IP against 3xFLAG affinity label


title: RNA-seq FTO mutant shoot rep2

description:
non-ribosomal RNA
shoot_gene_featureCounts.txt
shoot_repeat_featureCounts.txt

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

value: FTO homogeneous transgenic mutant

tag: ip antibody


title: set2-1_NETseq


title: ESC +pRNA -H3K9me3

data_processing:
Fastq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

value: Embryonic stem cells

tag: chip antibody

value: transfection with pRNA


title: rtt103-2_NETseq

value: RPB3::RPB3-3xFLAG NAT, RTT103::HIS3


title: RNA-seq FTO mutant shoot rep1

tag: tissue

value: shoot


title: rtt103-1_NETseq


title: m6A-seq FTO root IP rep2

description:
FTO_root_rep2_gene_MeTPeak.txt
FTO_root_rep2_repeat_MeTPeak.txt

source_name: FTO transgenic rice root

value: FTO homogeneous transgenic

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1617S)


title: genomic DNA from ovarian cancer tissues [G639]

description:
Ovarian tumor tissue
R03C01

tag: disease

value: ovarian cancer

tag: Sex

value: female

tag: Stage

tag: grade

tag: age

tag: tumour subtypes

value: serous

tag: platinum and drug treatment

value: carboplatin/pacelitaxel

tag: dls

tag: recurence date

tag: reccurrence within 5 years

value: Resistant


title: ESC +RNA Ctrl -H3K9me3

tag: cell line

tag: cell type

tag: treatment

value: transfection with RNA Ctrl


title: ESC +pRNA -H3K9me2


title: genomic DNA from ovarian cancer tissues [G684]

description:
Ovarian tumor tissue
R02C01

tag: tumour subtypes2

value: clear cell

tag: primary chemotherapy treatment

value: carboplatin+paclitaxel+avastin

tag: recurrence

tag: doot

tag: dodx

tag: survivial status


title: m6A-seq FTO root IP rep1

description:
FTO_root_rep1_gene_MeTPeak.txt
FTO_root_rep1_repeat_MeTPeak.txt

tag: ecotype

tag: genotype

tag: ip antibody

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1616S)

tag: tissue

value: root


title: rtf1-4_NETseq

tag: strain

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, RTF1::HIS3

tag: rna fraction

value: Nascent RNA

tag: ip

value: IP against 3xFLAG affinity label


title: ESC +RNA Ctrl -H3K9me2

tag: cell line

value: Embryonic stem cells

tag: chip antibody


title: rtf1-3_NETseq


title: genomic DNA from ovarian cancer tissues [G631]

description:
Ovarian tumor tissue
R03C01

tag: disease

tag: Sex

tag: Stage

tag: grade

tag: age

tag: tumour subtypes

tag: platinum and drug treatment

tag: dls

tag: recurence date

tag: reccurrence within 5 years

value: Sensitive


title: ESC input

tag: cell type

tag: treatment

value: transfection with pRNA


title: genomic DNA from ovarian cancer tissues [G633]

description:
Ovarian tumor tissue
R05C01

value: carboplatin/cyclophosphamide

tag: dodx

tag: survivial status


title: m6A-seq FTO shoot IP rep1

description:
FTO_shoot_rep1_gene_MeTPeak.txt
FTO_shoot_rep1_repeat_MeTPeak.txt

source_name: FTO transgenic rice shoot

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

value: FTO homogeneous transgenic

tag: ip antibody

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1614S)

tag: tissue

value: shoot


title: cla13: projection

city: Vancouver

first: Mark

middle: Steven

last: Cembrowski

state: BC

address: 2350 Health Sciences Mall

department: Cellular and Physiological Sciences

country: Canada

institute: University of British Columbia

data_processing:
Trim input file and remove adapters.
Tag reads with cell barcode and UMI
Align reads to PhiX, then to mouse.
Aggregate reads and de-duplicate UMIs.
Post-processing in R with custom scripts and Seurat.
Genome_build: NCBIM37
Supplementary_files_format_and_content: combinedTablesWt.txt: Counts table for WT mouse.
Supplementary_files_format_and_content: metadataWt.txt: Metadata for WT mouse
Supplementary_files_format_and_content: combinedTablesProj.txt: Counts table for projection dataset.
Supplementary_files_format_and_content: metadataProj.txt: Metadata for project dataset

source_name: Used for projection data

extract_protocol:
Microdissect, dissociate, and pool cells across acute brain slices in B6 mice. Some fluorophore-labeled projection neurons specifically targeted for RNA-seq, as noted in metadata
As in (Cembrowski et al., Cell, 2018)

value: Claustrum


title: m6A-seq FTO shoot IP rep2

description:
FTO_shoot_rep2_gene_MeTPeak.txt
FTO_shoot_rep2_repeat_MeTPeak.txt

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1615S)


title: cla12: projection


title: rtf1-2_NETseq

tag: strain

tag: genotype/variation

tag: rna fraction

tag: ip


title: rtf1-1_NETseq


title: rsc30-2_NETseq

value: RPB3::RPB3-3xFLAG NAT, RSC30::HIS3


title: genomic DNA from ovarian cancer tissues [G635]

description:
Ovarian tumor tissue
R07C01

tag: disease

value: ovarian cancer

tag: Sex

value: female

tag: Stage

tag: grade

value: high grade

tag: age

tag: tumour subtypes

value: serous

value: carboplatin/pacelitaxel

value: Resistant


title: cla11: projection


title: m6A-seq Nipp root IP rep1

description:
Nipp_root_rep1_gene_MeTPeak.txt
Nipp_root_rep1_repeat_MeTPeak.txt

source_name: WT rice root

value: wild type

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1612S)

value: root


title: m6A-seq Nipp root IP rep2

description:
Nipp_root_rep2_gene_MeTPeak.txt
Nipp_root_rep2_repeat_MeTPeak.txt

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1613S)


title: genomic DNA from ovarian cancer tissues [G642]

description:
Ovarian tumor tissue
R06C01

tag: platinum and drug treatment

tag: dls

tag: recurence date

tag: reccurrence within 5 years


title: rsc30-1_NETseq

value: Nascent RNA

value: IP against 3xFLAG affinity label


title: m6A-seq Nipp shoot IP rep2

description:
Nipp_shoot_rep2_gene_MeTPeak.txt
Nipp_shoot_rep2_repeat_MeTPeak.txt

source_name: WT rice shoot

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1611S)


title: cla10: projection

tag: tissue

value: Claustrum


title: cla9: projection


title: genomic DNA from ovarian cancer tissues [G630]

description:
Ovarian tumor tissue
R02C01

tag: dodx

tag: survivial status

value: Sensitive


title: rph1-2_NETseq

tag: strain

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, RPH1::HIS3

tag: rna fraction


title: m6A-seq FTO root input rep1

value: FTO homogeneous transgenic

tag: ip antibody


title: m6A-seq Nipp shoot IP rep1

description:
Nipp_shoot_rep1_gene_MeTPeak.txt
Nipp_shoot_rep1_repeat_MeTPeak.txt

value: anti-m6A (NEB, monoclonal,  rabbit,Cat. No. E1610S)

value: shoot


title: cla7: projection


title: cla8: projection


title: rph1-3_NETseq

tag: ip


title: genomic DNA from ovarian cancer tissues [G628]

description:
Ovarian tumor tissue
R08C01

tag: disease

value: ovarian cancer

tag: Sex

value: female

tag: Stage

tag: grade

value: serous


title: rph1-1_NETseq


title: ESC +RNA Ctrl -H3K9me3 repl2

tag: cell line

tag: antibody

tag: treatment

value: transfection with RNA Ctrl


title: cla6: projection


title: genomic DNA from ovarian cancer tissues [G626]

description:
Ovarian tumor tissue
R06C01

tag: age

tag: tumour subtypes

tag: platinum and drug treatment

value: carboplatin/taxol+avastin

tag: dls

tag: reccurrence within 5 years


title: genomic DNA from ovarian cancer tissues [G683]

description:
Ovarian tumor tissue
R02C01

tag: tumour subtypes2

value: clear cell

tag: primary chemotherapy treatment

value: carboplatin+paclitaxel

tag: recurrence

tag: doot

tag: dodx

tag: survivial status


title: rai1-1_NETseq

tag: strain

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, RAI1::HIS3

tag: rna fraction

value: Nascent RNA

tag: ip


title: genomic DNA from ovarian cancer tissues [G629]

description:
Ovarian tumor tissue
R01C01

tag: disease

tag: Sex

tag: Stage

tag: grade

value: carboplatin/pacelitaxel

tag: recurence date


title: m6A-seq FTO shoot input rep2

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

tag: ip antibody

tag: tissue


title: rad6-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, RAD6::HIS3

value: IP against 3xFLAG affinity label


title: rpb4-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, RPB4::HIS3


title: m6A-seq Nipp root input rep2

value: wild type

value: root


title: cla4: projection

value: Claustrum


title: cla5: projection


title: m6A-seq FTO shoot input rep1

value: FTO homogeneous transgenic

value: shoot


title: paf1-4_NETseq

value: RPB3::RPB3-3xFLAG NAT, PAF1::HIS3


title: genomic DNA from ovarian cancer tissues [G625]

description:
Ovarian tumor tissue
R05C01

value: ovarian cancer

value: female

tag: age

tag: tumour subtypes

value: serous

tag: platinum and drug treatment

tag: dls

tag: reccurrence within 5 years

value: Sensitive


title: m6A-seq Nipp root input rep1


title: cla3: WT mouse, no projection

source_name: WT mouse, no projection data


title: genomic DNA from ovarian cancer tissues [G627]

description:
Ovarian tumor tissue
R07C01

value: high grade

value: carboplatin/weekly pacelitaxel

tag: dodx

tag: survivial status


title: paf1-2_NETseq

tag: strain

tag: genotype/variation

tag: rna fraction

value: Nascent RNA

tag: ip


title: cla2: WT mouse, no projection


title: m6A-seq Nipp shoot input rep2

value: Nipponbare (Nipp)


title: m6A-seq Nipp shoot input rep1


title: genomic DNA from ovarian cancer tissues [G623]

description:
Ovarian tumor tissue
R03C01

tag: disease

tag: Sex

tag: Stage

tag: grade

value: carboplatin/pacelitaxel, tamoxifen


title: RNA-seq FTO root rep2

tag: ecotype

tag: genotype

tag: ip antibody


title: m6A-seq FTO root input rep2

tag: tissue


title: cla1: WT mouse, no projection


title: RNAseq of Arabidopsis: ddm1_rep3

city: Stockholm

first: Cheng

last: Zhao

state: Stockholm

address: H9 Klinisk vetenskap,171 77 Stockholm

department: Clinical Science

country: Sweden

institute: Karolinska institute

data_processing:
For RNA-seq data, low-quality sequences and adaptors were trimmed using Trimmomatic. Clean reads were mapped to the reference genome by TopHat with the parameter "-g 1". The total number of reads mapping to each gene was calculated with the htseq-count script in HTSeq with the parameter “--nonunique all”, to minimize over-estimation of TE expression caused by overlapping genes; read counts for each TE were calculated by htseq-count with parameters "--nonunique all  -m intersection-strict" based on the TE-only annotation. Genes and TEs with a normalized expression level of at least one count per million mapped reads (CPM) in three or more libraries were considered as expressed. Principle component analysis (PCA) based on the transcript levels of the expressed protein-coding genes and TEs was performed using the prcomp function in R software with default settings. Differentially expressed TEs with at least a 2-fold change in expression and an FDR < 0.05 were identified by the R package edgeR using the trimmed mean of M-values (TMM) method.
For methylation data, low-quality sequences and adapters were trimmed using Trimmomatic with parameters “LEADING: 3 SLIDINGWINDOW: 4: 30 MINLEN: 36”, and clean reads were mapped to the A. thaliana TAIR 10 genome using Bisulfite Sequence Mapping Program (BSMAP) with parameters “-v 2 -S 1” which allowing 2 mismatches. The methratio.py script from BSMAP with parameters “-r -z -p -m 1” was used to extract the methylation ratio from mapping results; only mapped reads after deduplication were considered for subsequent analyses.
For ChIP-seq data analysis, around 15.2 million raw paired-end reads were obtained for each sample and subsequently cleaned by Trimmomatic. Clean reads were mapped to the reference genome by Bowtie2 using the parameter "--very-sensitive --no-unal --no-mixed --no-discordant -k 2". Subsequently, uniquely mapped reads were selected and marked as duplicates using the Picard tool followed by using the SAMtools “rmdup” command. Coverage of deduplicated reads was normalized to 1 × sequencing depth by bamCoverage in deepTools with parameters "--normalizeUsing RPGC --exactScaling -bs 10".
Identification of non-reference TE insertions with TSDs (Target Site Duplications) was conducted using SPLITREADER with some modifications.
Genome_build: TAIR10
Supplementary_files_format_and_content: methylation wig files, RNA-seq coverage bedgGraph, ChIP-seq BigWig files

organism: Arabidopsis thaliana

source_name: seedling

growth_protocol: Arabidopsis seeds were plated on 1/2 Murashige and Skoog (MS) medium with 0.6% agar and 1.5% sucrose and stratified for 7 days at 4℃ in darkness before being transferred to the growth chamber (16 h light/8 h dark, 22℃)

extract_protocol:
Total RNA was extracted from two-week-old seedlings using the RNeasy Plant Mini kit (Takara)
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: age

value: two-week-seedling

value: seedling


title: RNA-seq FTO root rep1


title: paf1-1_NETseq

value: IP against 3xFLAG affinity label


title: RNAseq of Arabidopsis: ddm1_rep2


title: genomic DNA from ovarian cancer tissues [G622]

description:
Ovarian tumor tissue
R02C01

tag: tumour subtypes

tag: platinum and drug treatment

tag: dls

tag: recurence date


title: npl3-2_NETseq

tag: strain

value: RPB3::RPB3-3xFLAG NAT, NPL3::HIS3


title: InjuredSpinalCord_1wpi_KD, rep3

type: RNA

first: Oscar

last: Seira

address: 818 west 10th Avenue. Blusson Spinal Cord Center. ICORD

institute: University of British Columbia (UBC)/ICORD

hyb_protocol: The microarray was hybridized with the targets for 17 h at 65°C using a Gene Expression Hybridization Kit (Agilent Technologies), washed using Gene Expression Wash Buffers Pack (Agilent Technologies). All steps were performed according to the manufacturer (Agilent Technologies).

scan_protocol: Slides were scanned immediately after washing on the  and scanned by a Microarray Scanner (G2565CA, Agilent Technologies)

data_processing: The Feature Extraction Software v9.1.3.1 (Agilent Technologies) was used to extract and analyze the assay signals and subsequently determine the signal-to-noise ratios from the microarray images. Microarray raw data (.txt files) were imported into R v. 3.0.1 (Team RDC, 2012) and analyzed with the Bioconductor (Gentleman et al., 2004) packages Agi4×44PreProcess and limma (Smyth, 2005). Briefly, after quality check, the microarray probes were filtered and their median foreground intensity was normalized within and between arrays according to Agi4×44Preprocess and limma user guides, respectively. Finally, the probes were tested for differential expression using a linear model followed by Bayes moderated t-test (Smyth, 2005) for the comparisons of interest. Genes with nominal p < 0.05 were accepted to be differentially expressed.

organism: Rattus norvegicus

source_name: Spinal Cord Homogenates, 1wpi, KD

label_protocol: Whole-genome gene expression profiles of SCI rats treated with a CD (n=4) or KD (n=4) were measured using a SurePrint G3 Rat GE 8 X 60K Microarray ((G4858A-028279, Agilent Technologies, Palo Alto, CA).  A total of 200 ng of total RNA was used and labeled using the Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies) with Cy3.

extract_protocol: Gene expression 7 days after SCI surgery was chosen to identify and characterize changes between CD (n=4) and KD (n=4) treated rats. Total RNA was isolated from 5-mm segment centered on the site of impact according to the instructions of the supplier using the miRvana miRNA isolation kit (Ambion). RNA quantity and integrity were assessed by (Nanodrop ND-1000, Thermo Scientific, USA) and microfluidics-based electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, USA), respectively. RNA samples with OD 260/280 of approximately 2.0 and RIN ≥ 8.0 were used for microarray experiments.

treatment_protocol: A 5 mm segment of the ipsilateral side of the spinal cord centered on the impact site was removed for extraction of RNA. Total RNA was extracted using a Trizol Reagent (Invitrogen, CA, USA) according to the manufacturers’ manual.

value: Sprague Dawley

value: Left C5 Hemicontused Spinal Cord

tag: gender

value: male

tag: treatment

value: Ketogenic Diet (KD)


title: InjuredSpinalCord_1wpi_KD, rep4


title: RNA-seq FTO shoot rep1

value: FTO homogeneous transgenic

value: shoot


title: nap1-3_NETseq

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, NAP1::HIS3

tag: rna fraction

tag: ip


title: InjuredSpinalCord_1wpi_KD, rep2


title: genomic DNA from ovarian cancer tissues [G615]

description:
Ovarian tumor tissue
R03C01

tag: disease

value: ovarian cancer

tag: Sex

value: female

tag: Stage

value: serous

value: carboplatin/pacelitaxel

tag: dodx

tag: survivial status

tag: reccurrence within 5 years

value: Sensitive


title: InjuredSpinalCord_1wpi_SD, rep4

source_name: Spinal Cord Homogenates, 1wpi, SD

tag: tissue

value: Standard or Control Diet (SD)


title: genomic DNA from ovarian cancer tissues [G621]

description:
Ovarian tumor tissue
R01C01

tag: grade

tag: age

tag: tumour subtypes

value: carboplatin/weekly pacelitaxel


title: RNAseq of Arabidopsis: ddm1_rep1

tag: genotype

value: two-week-seedling

value: seedling


title: genomic DNA from ovarian cancer tissues [G624]

description:
Ovarian tumor tissue
R04C01

tag: platinum and drug treatment

tag: dls

tag: recurence date


title: InjuredSpinalCord_1wpi_KD, rep1

tag: strain

tag: gender

tag: treatment


title: RNAseq of Arabidopsis: ddm1nrpd1_rep2


title: genomic DNA from ovarian cancer tissues [G614]

description:
Ovarian tumor tissue
R02C01

tag: disease


title: genomic DNA from ovarian cancer tissues [G616]

description:
Ovarian tumor tissue
R04C01

tag: Sex


title: genomic DNA from ovarian cancer tissues [G617]

description:
Ovarian tumor tissue
R05C01

tag: Stage


title: nap1-1_NETseq

tag: genotype/variation

tag: rna fraction

value: Nascent RNA

tag: ip

value: IP against 3xFLAG affinity label


title: npl3-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, NPL3::HIS3


title: nap1-2_NETseq


title: genomic DNA from ovarian cancer tissues [G620]

description:
Ovarian tumor tissue
R08C01

tag: dodx

tag: survivial status

tag: reccurrence within 5 years


title: genomic DNA from ovarian cancer tissues [G619]

description:
Ovarian tumor tissue
R07C01

tag: grade

tag: age

tag: tumour subtypes


title: genomic DNA from ovarian cancer tissues [G618]

description:
Ovarian tumor tissue
R06C01

tag: platinum and drug treatment

tag: dls


title: RNA-seq Nipp root rep2

tag: ecotype

value: Nipponbare (Nipp)

tag: genotype

value: wild type

tag: ip antibody

tag: tissue

value: root


title: RNAseq of Arabidopsis: ddm1nrpd1_rep1


title: InjuredSpinalCord_1wpi_SD, rep3

tag: strain

value: Sprague Dawley

value: Left C5 Hemicontused Spinal Cord

tag: gender

value: male

tag: treatment


title: RNA-seq Nipp shoot rep2

value: shoot


title: leo1-4_NETseq

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, LEO1::HIS3

tag: rna fraction


title: RNA-seq Nipp root rep1


title: RNAseq of Arabidopsis: ddm1npre1_rep2


title: paf1-3_NETseq

value: RPB3::RPB3-3xFLAG NAT, PAF1::HIS3

tag: ip


title: RNA-seq Nipp shoot rep1


title: RNAseq of Arabidopsis: ddm1npre1_rep3


title: INS1 832/13 RNA_rep3

city: Ann Arbor

first: Stephen

middle: C. J.

last: Parker

state: MI

address: 100 Washtenaw Ave

department: Computational medicine and Bioinformatics

institute: University of Michigan

description: We transfected the STARR-seq plasmid library into the rat 832/13 cell line, collected the RNA 24 hours, prepared and sequenced the library

data_processing:
Library strategy: STARR-seq
Quantifying Barcode counts from STARR-seq: The STARR-seq barcode sequencing data included the input DNA barcode library along with three cDNA barcode libraries representing three biological replicated. We processed this data through a custom pipeline which quantified barcode counts while accounting for sequencing errors. We extracted the DNA barcodes from the input DNA library (first 16 bp of the read-1 fastq file) and clustered these at an edit distance of 0 followed by computing the DNA counts for each read group of DNA sequencing files. We then aggregated the read groups and collapsed counts for barcodes using the sequence clustering algorithm Starcode (https://github.com/gui11aume/starcode) [DOI: 10.1093/bioinformatics/btv053]. We repeated this process for the cDNA barcode counts for each replicate, with the added step of removing PCR-duplicated barcodes using the UMI information (UMI sequence was the reverse complement of the first 6bp of the read-2 fastq file). The pipeline is shared at https://github.com/ParkerLab/STARR-seq-Analysis-Pipeline.
Tetsing for CAGE element transcriptional activity: We matched the barcodes with CAGE inserts using results from CAGE insert-barcode pairing experiment (Supplementary data “cage_insert_barcode_pairing.txt”). We first retained barcodes with at least 10 DNA counts, and further retained CAGE elements that had at least two such qualifying barcodes. This was the set of N=3,446 CAGE elements quantifiable in our assay. To quantify STARR-seq activities from these count-based data, we used the tool MPRAnalyze (version 1.3.1) (https://github.com/YosefLab/MPRAnalyze}) [DOI 10.1101/527887] that models DNA and RNA counts in a negative binomial generalized linear model. This approach is more robust than using metrics such as the aggregated ratio, which is the ratio of the sum of RNA counts across barcodes divided by the sum of DNA counts across barcodes and loses the statistical power provided by multiple barcodes per tested element; and the mean ratio, which is the mean of the observed RNA/DNA ratios across barcodes which can be quite sensitive to low counts and noise. We corrected for library depth for the three replicates using upper quartile normalization via the ‘estimateDepthFactors’ function in MPRAnalyze. STARR-seq activity is quantified by estimating the transcription rate for each element in the dataset, followed by identifying active elements that induce a higher transcription by testing against a null. MPRAnalyze fits two nested generalized linear models - the DNA model estimates plasmid copy numbers, and the RNA model is estimates transcription rate. We included barcode information in the DNA model which allows different estimated counts for each barcode, and increases the statistical power of the model. Replicate information was included in the RNA model. MPRAnalyze then tests the transcriptional activity of each element against a null distribution and computing Z and Median-Absolute-Deviation (MAD) scores. The null is based on the assumption that the mode of the distribution of transcription rate estimates is the center of the null distribution, and that values lower than the mode all belong to the null. Thus, values lower than the mode are used to estimate the variance of the null.
Genome_build: not applicable since the synthetic barcode sequences and not mapped to any genome
Supplementary_files_format_and_content: File "cage_insert_barcode_pairing.tsv" is a tab separated values (tsv) files containing the hg19 coordinates of the CAGE elements tested in the STARR-seq experiment and the corresponding barcode sequences. These sequenced barcodes represent the STARR-seq experiment readout. File "cage_insert_starrseq_counts_filtered.tsv" includes CAGE elements hg19 coordinates, corresponding barcode sequences, the DNA count and the RNA counts for the three replicates for the barcode sequences. CAGE elements in this file are what were selected for statistically testing the STARR-seq transcriptional activity. Specifically, we selected barcodes that had a minimum of 10 DNA counts, and selected CAGE elements that had minimum 2 such qualifying barcodes.

source_name: Rat INS1 832/13 cell line

extract_protocol:
We electroporated 10 ug of barcoded STARR-seq library into 25 million the 832/13 rat insulinoma cell line for each biological replicate (3 replicates), and harvested the cells twenty-fours later. We isolated total RNA using TRIZOL reagent (Life Technologies) following the manufacturer’s protocol up to phase separation. After phase separation, we transferred the aqueous phase of the solution to a new 1.5 mL Eppendorf tube, added 1:1 volume of 100\% ethanol, and then column-purified using the Direct-zol RNA Miniprep Kit (Zymo) following the manufacturer’s protocol. We further purified mRNA using Dynabeads oligo(dT) beads (Thermo Fisher). We treated 2 ug of mRNA with RNase-free DNaseI (invitrogen) to eliminate possible plasmid DNA contamination, and then reverse transcribed it into cDNA using the SuperScript III First-Strand Synthesis kit (invitrogen) with a custom primer that specifically recognizes ‘STARR transcripts’ (i.e. mRNA that had been transcribed from the STARR-seq plasmids). To eliminate any residual plasmid contamination in cDNA samples, we treated cDNA with DpnI (NEB), and purified the reaction using the DNA Clean and Concentrator-5 kit (Zymo).
We constructed Illumina sequencing library via two serial rounds of PCR. In the first round, we used a primer set to specifically amplify STARR transcripts using cDNA as starting material. In the second round, we used a primer set to append the P5/P7 Illumina sequences using the PCR product from the first round as starting material. In both rounds, we PCR-amplified the fragments until the amplification curve reached a mid-log phase, and then purified the products for subsequent steps using the DNA Clean and Concentrator-5 kit (Zymo). To identify the barcodes corresponding to each CAGE element in the STARR-seq plasmid, 1ng of each library constructed was used in a polymerase chain reaction with primers flanking the allele and the barcode to generate fragments which were subsequently gel verified and extracted using Zymo gel extraction kit (Zymo). 25ng of the purified product was subjected to self-ligation at 16 °C overnight in a total volume of 50uls and column purified using Qiagen (Qiagen) PCR purification kit as per manufacturers recommendations. The purified fragments were subsequently treated with 10U of Plasmid-Safe ATP-Dependent DNase for 1 hour in the presence of 25mM ATP to remove unligated linear DNA fragments. 1ul of the recircularized fragments were subjected to another round of PCR resulting in a smaller fragment. Briefly an aliquot of this product was diluted 1:10 in DNAse/RNase free water and amplified in a PCR reaction, with Illumina P5 and P7 adapters, until saturation to generate libraries. The libraries were subsequently column purified, quantified and sequenced.

tag: cell line


title: 4C-rDNA K562 rep1

city: Moscow

first: Nickolai

last: Tchurikov

address: 32, Vavilova str.

department: Department of Epigenetic Mechanisms of Gene Expression Regulation

country: Russia

institute: Engelhardt Institute of Molecular Biology

description: Replica 1

data_processing:
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control.
Sequenced reads were trimmed for adaptor sequences and low quality sequences by cutadapt v. 2.7 using the following options: --trim-n --times=4 --minimum-length 20 -q 26 --untrimmed-output=4C_notail.fastq.gz -g GCCTAAGCCTGCTGAGAACTTTC -g CAGCATTCTGTAGGGAGATCAAATC -a GAAAGTTCTCAGCAGGCTTAGGC -a GATTTGATCTCCCTACAGAATGCTG
Untrimmed sequences from the file 4C_notail.fastq.gz were trimmed for the following adaptor sequences by cutatapt v. 2.7 using the following options --trim-n --times=4 --trimmed-only --minimum-length 20 -q 26 -g TCTTTGAAAAAAATCCCAGAAGTGGT -g AAGTCCAGAAATCAACTCGCCAGT -a ACTGGCGAGTTGATTTCTGGACTT -a ACCACTTCTGGGATTTTTTTCAAAGA. Then outputs of previous and current adaptor removal steps were concatenated in one file 4C_trimmed.fastq.gz for furher processing
Trimmed reads were mapped to hg19p13 by bwa 0.7.17-r1188 using the mem algorithm. All non-aligned reads were removed from resulting alignment file by samtools 1.6: -b -S -F 4 -@ 10 allalign.sam > aligned.bam
Mapping and alignment results were converted to resulting tables using samtools 1.6 and ad hoc Perl scripts.
Each replica results table was converted to bedGraph file for further processing using reads amount as data value. Intersection file was built by bedtools v. 2.29.1 with the following command line: bedtools intersect -a 4C_K562_hg19.rep1.bedGraph -b 4C_K562_hg19.rep2.bedGraph -wb. Ad hoc Perl script was used to convert bedtools output file back to bedGraph file with summing up in the overlapped regions.
All data processing steps were performed for H.Sapiens hg38p12 build too.
Genome_build: Homo Sapiens hg19/GRCh37.p13 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Genome_build: Homo Sapiens hg38/GRCh38.p12 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Supplementary_files_format_and_content: Tab-delimited text file include the following features of each mapping: begin, end, length, coverage, number of reads, sequence. bedGraph files for each replica and intersection are supplied too.

source_name: blood

growth_protocol: K562 cells were grown in RPMI with 10% FCS + 2 mM glutamine in a humidified incubator at 37°C and 5% CO2 to 80% confluency.

extract_protocol:
DNA samples were prepared as described before (Dekker et al., 2002; Osborne et al., 2004; http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3978).  Cross-linking  To suspension containing about 20x106 K562 cells in 40 ml of DMEM formaldehyde solution was added to final concentration 1.5%. After mixing incubation of cell suspension was performed for 10 min at room temperature with mixing. The quenching with 2.75 ml of 2M glycin (final concentration 0.125M) was performed. After incubation at room temperature for 5 min the suspension was cooled for 15 min in ice bath and then cells were collected by centrifugation for 15 min at 3500 rpm at +2°C.  Lysis   The pellet of cells was resuspended at 0°C in 1ml of buffer containing 10 mM tris-HCl buffer, pH 8, 10 mM NaCl, 0.2% NP-40 and freshly added protease inhibitors – 0.1 mM PMSF and 1:500 protease inhibitor cocktail (Sigma). After incubation for 15 min cells were homogenized by passing through insulin syringe about 50 times into Eppendorf. Then nuclei were spin down by centrifugation for 5 min at 5000 rpm in Eppendorf centrifuge 5415 R at +2°C.  Digestion with EcoRI  The nuclei pellet was resuspended in 756 ul of solution containing 40 mM tris-HCl buffer, pH 7.4, 50 mM NaCl, 10 mM MgCl2 and 10 mM 2-mercaptoethanol. Then 20% SDS was added to final concentration 0.3% and incubation with shaking was performed for 1 h at +37°C. To sequester SDS 180 l of 10% Triton X-100 was added and the incubation for 1 h at +37°C was performed. Before digestion 1 l of BSA (5 mg/ml) was added with mixing. Then 50 l of EcoRI (10 u/l) was added and after mixing digestion was performed overnight at +37°C.   Ligation  To inactivate the restriction enzyme, 35 l of 20% SDS was added (final concentration 0.7%) and the probe was heated to 65°C for 30 min. Then the mixture was transferred into 15 ml Nunc tube and consequently 375 l of 20% Triton X-100 (to final concentration 1%), 750 l of 10x ligase buffer, 7,5 l of BSA (5 mg/ml), 80 l of 100 mM ATP, and 5241 l of milliQ water  were added and the final 7.5 ml solution was well mixed. Then 10 l of T4 DNA ligase (200 u/l) was added and after mixing incubation was performed for 5 h at +16°C and then for 30 min at room temperature. Usually during ligation DNA concentration was equal to 2 ng/l.    DNA purification  For isolation of DNA 50 l of proteinase K (10 mg/ml) was added (final concentration 50 g/ml) and after mixing incubation was performed at 55°C B1overnight. For RNA digestion 40 l of RNase A (10 mg/ml) was added (final concentration 0.5 g/ml) and after mixing incubation was performed for 30 min at +37°C. After extensive extraction with phenol-chloroform extraction (three times with 7 ml each) DNA was precipitated by 2.5 vol. of ethanol after addition of 40 l 10 mg/ml glycogen and 175 l 4M NaCl. The final DNA pellet was washed twice with 70% ethanol and then dissolved in 0.1xTE.   Digestion with FaeI  About 15 g of DNA was digested in 250 ul solution with 75 u of FaeI overnight at +37°C. Then the enzyme was inactivated by heating at 65°C for 30 min. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 100 l of 0.1xTE.   Ligation  For circularization 15 g of DNA was incubated in 8 ml of T4 DNA ligase buffer containing 400 u of T4 DNA ligase for 5 h at +16°C. DNA was isolated after phenol-chloroform extraction, precipitated by ethanol and dissolved in 50 l of 0.1xTE.   PCR and library preparation  For preparation of 4C-rDNA libraries about 30 ng of 4C DNA preparations were used for 35 cycles of PCR with primers 5’ TCTTTGAAAAAAATCCCAGAAGTGGT 3’ and 5’ AAGTCCAGAAATCAACTCGCCAGT 3’ for each biological replicate. Deep sequencing of libraries were performed using HiSeq1500 (Illumina) using up to 250-nt long reads.
For preparation of 4C-rDNA libraries about 30 ng of 4C DNA preparations were used for 35 cycles of PCR with primers 5’ TCTTTGAAAAAAATCCCAGAAGTGGT 3’ and 5’ AAGTCCAGAAATCAACTCGCCAGT 3’ for each biological replicate. Deep sequencing of libraries were performed using HiSeq1500 (Illumina) using up to 250-nt long reads.

value: blood

tag: disease state

value: erythroleukemia


title: RNA-seq FTO shoot rep2

value: FTO homogeneous transgenic


title: 4C-rDNA K562 rep2

description: Replica 2


title: RNAseq of Arabidopsis: ddm1npre1_rep1

value: two-week-seedling


title: leo1-2_NETseq


title: INS1 832/13 RNA_rep2


title: leo1-1_NETseq


title: InjuredSpinalCord_1wpi_SD, rep1

value: Standard or Control Diet (SD)


title: INS1 832/13 RNA_rep1


title: isw2-4_NETseq

value: RPB3::RPB3-3xFLAG NAT, ISW2::HIS3

value: Nascent RNA


title: genomic DNA from ovarian cancer tissues [G612]

description:
Ovarian tumor tissue
R08C01

tag: disease

value: ovarian cancer

tag: Sex

value: female

tag: Stage

tag: grade

value: serous

value: carboplatin/pacelitaxel

tag: dodx

tag: survivial status

tag: recurence date

tag: reccurrence within 5 years

value: Sensitive


title: InjuredSpinalCord_1wpi_SD, rep2

value: Sprague Dawley

tag: tissue

value: Left C5 Hemicontused Spinal Cord


title: genomic DNA from ovarian cancer tissues [G571]

description:
Ovarian tumor tissue
R06C01

tag: age

tag: tumour subtypes

tag: platinum and drug treatment

tag: dls

value: Resistant


title: leo1-3_NETseq

tag: strain

tag: genotype/variation

value: RPB3::RPB3-3xFLAG NAT, LEO1::HIS3

tag: rna fraction

value: IP against 3xFLAG affinity label


title: RNAseq of Arabidopsis: ddm1nrpd1_rep3

tag: genotype

value: seedling


title: 4C-rDNA hESM01 rep2

description: Replica 2

data_processing:
Base calling and quality control were performed in real time with standard Illumina analysis pipeline using a phiX control.
Sequenced reads were trimmed for adaptor sequences and low quality sequences by cutadapt v. 2.7 using the following options: --trim-n --times=4 --minimum-length 20 -q 26 --untrimmed-output=4C_notail.fastq.gz -g GCCTAAGCCTGCTGAGAACTTTC -g CAGCATTCTGTAGGGAGATCAAATC -a GAAAGTTCTCAGCAGGCTTAGGC -a GATTTGATCTCCCTACAGAATGCTG
Untrimmed sequences from the file 4C_notail.fastq.gz were trimmed for the following adaptor sequences by cutatapt v. 2.7 using the following options --trim-n --times=4 --trimmed-only --minimum-length 20 -q 26 -g TCTTTGAAAAAAATCCCAGAAGTGGT -g AAGTCCAGAAATCAACTCGCCAGT -a ACTGGCGAGTTGATTTCTGGACTT -a ACCACTTCTGGGATTTTTTTCAAAGA. Then outputs of previous and current adaptor removal steps were concatenated in one file 4C_trimmed.fastq.gz for furher processing
Trimmed reads were mapped to hg19p13 by bwa 0.7.17-r1188 using the mem algorithm. All non-aligned reads were removed from resulting alignment file by samtools 1.6: -b -S -F 4 -@ 10 allalign.sam > aligned.bam
Mapping and alignment results were converted to resulting tables using samtools 1.6 and ad hoc Perl scripts.
Each replica results table was converted to bedGraph file for further processing using reads amount as data value. Intersection file was built by bedtools v. 2.29.1 with the following command line: bedtools intersect -a 4C_hESM01_hg19.rep1.bedGraph -b 4C_hESM01_hg19.rep2.bedGraph -wb. Ad hoc Perl script was used to convert bedtools output file back to bedGraph file with summing up in the overlapped regions.
All data processing steps were performed for H.sapiens hg38p12 build too.
Genome_build: Homo sapiens hg19/GRCh37.p13 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Genome_build: Homo sapiens hg38/GRCh38.p12 genome with included to chr14 from address 1 Human ribosomal DNA U13369
Supplementary_files_format_and_content: Tab-delimited text file include the following features of each mapping: begin, end, length, coverage, number of reads, sequence. bedGraph files for each replica and intersection are supplied too.

source_name: blastocyst

growth_protocol: hESM01 were cultured in medium consisted of 80% KnockOut DMEM, 20% FBS, 1 mM glutamine, 1% nonessential amino acids, 50 units/ml penicillin, 50 μg/ml streptomycin (all from Invitrogen), 0.1 mM b-mercaptoethanol (Sigma), and 4 μg/ml bFGF (Chemicon) (40 Gy) on mitomycin C  treated (10 μg/ml, Sigma) mouse embryonic fibroblasts (MEF) on tissue culture dishes precoated with 0.1% gelatin (Merck). MEFs were prepared from day 12.5 p/c fetuses of F1 (C57BL/6J x CBA/Ca) mice. hESM01 colonies were replated by exposure to type IV collagenase (200U/ml, Invitrogen) and mechanical dissociation every 5–6 days. Cell lines from 20 to 23 passages were used for the experiments.

tag: cell line

value: hESM01 line

value: embryo

tag: disease state

value: wild type


title: genomic DNA from ovarian cancer tissues [G572]

description:
Ovarian tumor tissue
R07C01

tag: disease

tag: Sex

tag: Stage


title: BSseq of Arabidopsis: nrpe1_rep1

extract_protocol:
Genomic DNA was extracted from two-week-old seedlings using the DNeasy Plant Maxi kit (Qiagen).
Bisulfite treatment, library construction, and sequencing were performed at the PSC Genomics Core Facility.

value: two-week-seedling


title: genomic DNA from ovarian cancer tissues [G613]

description:
Ovarian tumor tissue
R01C01

tag: grade

value: carboplatin/taxol+avastin

tag: dodx

tag: survivial status


title: S3_2

city: Hong Kong

first: Andrew

middle: M.

address: Room 407, 4/F, Lo Kwee-Seong Integrated Biomedical Sciences Building,, Area 39, The Chinese University of Hong Kong

department: School of Biomedical Science

institute: The Chinese University of Hong Kong

data_processing:
Mapped by Bowtie2
Quantified by RSEM
Differential analysis by PossionDis algorithm
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

growth_protocol: Cells propagated in 10% FBS and passaged when reached ~80% confluence

extract_protocol:
Total RNA was extracted using an RNA extraction kit (Omega Bio-Tek)
BGI reagent

tag: cell type

value: Schwannian-like cells

tag: type

tag: passages

value: Passage 35

value: ALK F1174L mutation


title: isw2-3_NETseq

value: RPB3::RPB3-3xFLAG NAT, ISW2::HIS3

value: Nascent RNA

tag: ip


title: BSseq of Arabidopsis: nrpd1_rep2

tag: age

tag: tissue


title: isw2-2_NETseq

tag: strain

tag: genotype/variation


title: BSseq of Arabidopsis: ddm1_rep2

tag: genotype


title: 4C-rDNA hESM01 rep1

description: Replica 1

tag: cell line


title: NC3_1

value: Neuroblastic tumor cells


title: genomic DNA from ovarian cancer tissues [G453]

description:
Ovarian tumor tissue
R04C01

value: ovarian cancer

value: female

tag: tumour subtypes

value: serous

tag: platinum and drug treatment

value: carboplatin/pacelitaxel

tag: dls

tag: recurence date

tag: reccurrence within 5 years


title: isw1-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, ISW1::HIS3

tag: rna fraction

value: IP against 3xFLAG affinity label


title: BSseq of Arabidopsis: ddm1npre1_rep1

value: seedling


title: BSseq of Arabidopsis: nrpd1_rep1


title: ino80-3_NETseq

value: RPB3::RPB3-3xFLAG NAT, INO80::HIS3


title: isw1-2_NETseq


title: BSseq of Arabidopsis: ddm1nprd1_rep2


title: BSseq of Arabidopsis: ddm1_rep1


title: S3_1


title: NC3_2


title: BSseq of Arabidopsis: nrpe1_rep2


title: isw2-1_NETseq


title: BSseq of Arabidopsis: ddm1npre1_rep2


title: GNS166 shL2A exp3

city: Donostia

first: Jaione

last: Auzmendi

address: Paseo Dr. Beguiristain s/n

country: Spain

institute: IIS Biodonostia

data_processing:
NOT PROVIDED; REQUESTED
Genome_build: human GRCh38
Supplementary_files_format_and_content: geneDE_results.csv

source_name: GNS166 shL2A

growth_protocol: Glioma Neural Stem 166 (GNS166) cell line was cultured in adhesion in treated culture plates, in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) media, supplemented by 2 µg/mL laminin, 1% N2, 2% B27, 1.34% D-(+)-Glucose solution 45%, 2 mM L-Glutamine, 100 U/mL penicillin, 100 µM/mL streptomycin, 20 ng/ml basic fibroblast growth factor and 20 ng/ml epidermal growth factor. For splitting GNS166, accutase was used, with incubations of 2-5 minutes at 37ºC. DPBS was used for cell wash.

extract_protocol:
mRNA samples were extracted by TRIzol technique.
For library construction, RNA was extracted from a sample first. After DNase I treatment and performing quality control (QC), 200 ng of high-quality total RNA was proceed to library construction. Magnetic beads with Oligo (dT) are used to isolate mRNA. The mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer after adding dNTPs, RNase H and DNA polymerase I. Short fragments are purified and resolved with EB buffer for end repair and single nucleotide A (adenine) addition. After that, the short fragments are connected with sequencing adapters. The double-stranded cDNA library is completed through size selection and PCR enrichment. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. Lastly, the qualified RNA-seq libraries are sequenced using Illumina NovaSeq6000 in CD Genomics (Shirley, NY) after pooling according to its effective concentration and expected data volume.

treatment_protocol: LAMP2A was downregulated by lentiviral infections of short-hairpin RNA. Cells were incubated for 5 days after infection due to half-life of the protein. Infected cells were GFP positive.

value: Patient-derived glioma stem cells

tag: condition

value: LAMP2A downregulation


title: GNS166 shL2A exp1

value: Glioma Neural Stem 166 (GNS166) cell line

tag: cell type


title: BSseq of Arabidopsis: ddm1nprd1_rep1

value: two-week-seedling


title: ino80-4_NETseq


title: BSseq of Arabidopsis: ddm1cmt2_rep1


title: ARPE-19_TGF-β1 treated_rep3

city: Shanghai

first: Shuai

last: Yang

address: 301 Middle Yanchang Road

department: Department of Ophthalmology

country: China

institute: Shanghai Tenth People's Hospital, Tongji University School of Medicine

description: Gene expression in ARPE-19 treated with 10 ng/ml TGF-β1 for 48 h

hyb_protocol: 1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60°C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.

scan_protocol: The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).  Scan Area (61 x 21.6 mm)  Scan resolution (μm):5. Dye channel: Green.Green PMT:    XDR Hi 100%   XDR Lo 10%

data_processing: Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 3 out of 6 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis.

source_name: ARPE-19 cells

label_protocol: Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3' bias utilizing a mixture of oligo(dT) and random primers (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.

growth_protocol: Human RPE cell line ARPE-19 cells were obtained from the Eye Institute of Tongji University. Cells were cultured in DMEM/F12 culture media (Gibco, Life Technologies) with 10% fetal bovine serum (FBS) (Gibco, Life Technologies) at 37 °C in a humidified incubator containing 5% CO2. The culture media were change every 2–3 days. For microarray experiments, cells were trypsinized and seeded in 6 well plates and cultured for 12 h. Thereafter, cells were starved for 16 h.

extract_protocol: Total RNAs were extracted at indicated times with a TRIzol reagent kit.

treatment_protocol: After starved for 16 h, 10 ng/ml TGF-β1 (Invitrogen, Life Technologies) or dissolvent were added to ARPE-19 cells

tag: treatment

value: 10 ng/ml of TGF-β1 for 48 h


title: ino80-1_NETseq


title: GNS166 pGK exp3

source_name: GNS166 pGK

value: control


title: NGP_I15_3

first: Ruben

last: Dries

address: 360 Longwood Ave

department: Computational Biology

institute: DFCI

hyb_protocol: Following fragmentation, 12.5 ug of aRNA were hybridized at 45C for 16 hr at 60RPM on GeneChip Arrays (PrimeView). GeneChips were washed and stained in the Affymetrix Fluidics Station 450 according to the manurfacturer's instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.

scan_protocol: GeneChips were scanned using the GeneChip Scanner 3000 and images were extracted with Affymetrix GeneChip Expression Console.

data_processing: Microarray data were analyzed using a custom CDF file (GPL16043) that contained the mapping information of the ERCC probes used in the spike-in RNAs. The data were analyzed with Bioconductor using the MAS5 normalization method. Briefly, all chip data were imported in R (version 3.1.3) using the affy package, converted into expression values using the expresso command, normalized to take into account the different numbers of cells and spike-ins used in the different experiments and renormalized using loess regression fitted to the spike-in probes.

label: biotin

source_name: NGP Crispr edited cells

label_protocol: Biotinylated aRNA was prepared according to the standard Affymetrix protocol from 100 ng of total RNA (GeneChip 3' IVT Express Kit User Manual, Affymetrix).

growth_protocol: NGP CRISPR control cells and NGP ALK(LF655del) cells were grown in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin

extract_protocol: Biological triplicates (equivalent to 8 x 10e6 cells per replicate) were collected and homogenized in 1ml of TRIzol Reagent (Ambion), purified using the mirVANA miRNA isolation kit (Ambion) following the manufacturer’s instructions and re-suspended in nuclease-free water (Ambion). Total RNA was spiked-in with ERCC RNA Spike-In Mix (Ambion), treated with DNA-free DNase I (Ambion).

treatment_protocol: Cells were not treated with any compound before performing RNA extraction.

value: NGP ALK(LF655del) cells

value: Neuroblastoma NGP cells


title: ARPE-19_TGF-β1 treated_rep1


title: GNS166 pGK exp2


title: BSseq of Arabidopsis: Col_rep2

value: Col


title: NGP_I15_2


title: htz1-1_NETseq

source_name: Yeast

value: RPB3::RPB3-3xFLAG NAT, HTZ1::HIS3

value: Nascent RNA


title: NGP_I15_1


title: BSseq of Arabidopsis: Col_rep1


title: ARPE-19_TGF-β1 treated_rep2


title: ARPE-19_Control_rep2

description: Gene expression in normal ARPE-19

value: dissolvent of TGF-β1 for 48 h


title: ARPE-19_Control_rep3

description: Gene expression in normal ARPE-19


title: BSseq of Arabidopsis: cmt2_rep1


title: GNS166 pGK exp1


title: cdj-2

city: wuhan

first: li

last: dong

address: hongshan

institute: Huazhong University of Science and Technology

data_processing:
The libraries were sequenced on Illumina novaseq 6000 Platform for 6G raw data, and generated 150nt pair-end reads.
Raw data (raw reads) of FASTQ format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated
UMI sequences on each read were identified with UMI-tools (1.0.0), and reads with UMIs were subjected to subsequent analysis. To identified the duplicated reads, UMIs were firstly removed from the UMI reads, and remained parts of each reads were mapped to the reference genome using Hisat2.
Index of the reference genome was built using HISAT2 v2.0.4 and paired end clean reads were aligned to the reference genome using HISAT2 v2.0.4
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq R package (1.10.1)
Genome_build: mus_musculus_Ensembl_97
Supplementary_files_format_and_content: fpkm

source_name: fibroblast cell lines

growth_protocol: NIH3T3 cells were purchased from Wuhan University Preservation Center (Wuhan, China). All cells were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma), 50 U/mL penicillin, and 50 g/mL streptomycin (Gibco, cat. no.15070) at 37 °C under 5% CO2.

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


title: hpc2-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, HPC2::HIS3


title: GNS166 shL2A exp2


title: cdj-1


title: ino80-2_NETseq


title: hpc2-2_NETseq


title: X44_LCP0021_B

city: Bethesda

first: Amanda

last: Craig

state: Maryland

address: 37 Convent Dr. Bld.37/Rm.3044

institute: National Institutes of Health

data_processing:
Reads of the samples were trimmed for adapters and low-quality bases using Cutadapt [1] before alignment with the reference genome (Human - hg38) and the annotated transcripts using STAR [2]. Mapping statistics were calculated using Picard (2.18.26). Quantification was performed with RSEM [3]. Normalized counts were log transformed for downstream analysis.
Genome_build: HUMAN - hg38
Supplementary_files_format_and_content: csv; Gene expression matrix

source_name: Liver

extract_protocol: RNA was extracted from 58 total samples. Library preparation and sequencing were performed at the Clinical Cancer Research Sequencing Facility at the Frederick National Laboratory for Cancer Research, NCI. Libraries were constructed using TruSeq Stranded Total RNA Library Prep using the manufacturer’s recommendations. Sequencing was performed on 150bp paired end reads on an Illumina NovaSeq platform.  Libraries for four samples with low RNA concentration were constructed using NEBnext Ultra Low Input Total RNA Prep using the manufacturer’s recommendations. Sequencing was performed on 75bp paired end reads on an Illumina NextSeq platform.

tag: tag

value: Non-tumor


title: cdj-3


title: DNAseq of Arabidopsis: ddm1cmt2_rep1

source_name: rosette leaf

growth_protocol: Arabidopsis seeds were plated on 1/2 Murashige and Skoog (MS) medium with 0.6% agar and 1.5% sucrose and stratified for 7 days at 4℃ in darkness before being transferred to the growth chamber (16 h light/8 h dark, 22℃). Then, the 2-week-old seedlings were transplanted to soil for growth 1 week in growth chamber (16 h light/8 h dark, 22℃).

extract_protocol:
Genomic DNA was extracted from rosette leaf of three-week-old plants using the DNeasy Plant Maxi kit (Qiagen).
Library construction and sequencing were performed at the PSC Genomics Core Facility.

value: three-week-seedling

value: rosette leaf


title: hda1-6_NETseq

value: RPB3::RPB3-3xFLAG NAT, HDA1::HIS3

tag: ip

value: IP against 3xFLAG affinity label


title: NGP_control_3

source_name: NGP cells

value: NGP CRISPR control cells


title: Naïve-mouse-2

city: Ness Ziona

first: Inbar

last: Cohen Gihon

address: Reuven 24

department: Biochemistry

country: Israel

institute: Israel Institute For Biological Research

data_processing:
RTA (Illumina) for base calling
bcl2fastq2 (version 2.20) for converting BCL to fastq format, coupled with adaptor trimming.
Reads were mapped  to the reference genome Mouse: UCSC/mm10 using STAR(2.5.2b) and featureCounts(v1.5.0-p3)
Reads were mapped  to the reference genome Yersinia pestis CO92 (NC_003143,NC_003131, NC_003132, NC_003134) using bowtie2 (2.2.0) and htseq-count 0.6.1
Supplementary_files_format_and_content: Matrix table with raw read counts for every gene and every sample

source_name: Naïve mouse lungs

growth_protocol: Yersinia pestis Kimberly53 strain were used for intranasal infection. Bacterial cultures were grown at 28◦C overnight in HIB (BD, MD USA), supplemented with 0.2% (+) xylose and 2.5 mM CaCl2 (Sigma-Aldrich). The bacteria were used for intranasal infection of mice (OF1 strain).

extract_protocol:
One, 24 and 48 hours post infection, mice were sacrificed, lungs were removed and used for RNA extraction. RNA was also extracted from bacteria grown in culture (at 28° and 37° C) and naive mice, as control. Total RNA was extracted using the RNeasy kit (Qiagen) and residual DNA was digested using RNase-free DNase (Qiagen). In order to enrich the mRNA bacteria grown in culture (In vitro) we used riboZiro rRNA removal kit for depletion of rRNA, in each sample and ribozero gold rRNA depletion for dual RNA samples (mouse and Y. pestis).
Library construction using Illumina TruSeq chemistry

treatment_protocol: Mice (OF1)  were infected intranasally with 800,000 cfu/mouse (~1500 LD50).

value: OF1 strain

value: Naïve mouse


title: hda1-5_NETseq


title: NGP_control_2


title: htz1-2_NETseq


title: DNAseq of Arabidopsis: ddm1-1_rep2

tag: age


title: DNAseq of Arabidopsis: nrpd1_1_rep1

tag: tissue


title: X41_LCP0020_B


title: X56_LCP0026_B


title: DNAseq of Arabidopsis: ddm1-1_rep1


title: ARPE-19_Control_rep1

description: Gene expression in normal ARPE-19


title: Man1a2 homozygous dead placebo liver [PND_Liver_SIN238A9_S4]

city: La Jolla

first: Kim

last: Ngo

state: CA

address: 9500 Gilman Drive #0412

department: Department of Bioengineering

institute: University of California, San Diego

data_processing:
Illumina Novaseq reads were demultiplexed and were trimmed to filter out low-complexity reads, polyA tails, N runs
High-quality reads were then aligned to Ensembl mouse reference genome (Mouse.B38) using Omicsoft Sequence Aligner (OSA) on ArrayStudio (version 10.1), with index built to Ensembl genome for cDNA and ncRNA genome sequences.
Uniquely mapped and quantified transcript abundances from OSA output were imported into R software (version 4.0.3) for downstream differential gene expression analysis. Differential expression analysis of raw count data was performed using edgeR package (version 3.30.3) and differentially regualted genes in NAC-treated offspring were identified using two criteria: under the exactTest with dispersion set to biological coefficient of variation (bcv) to 0.2 method and absolute log2 fold-change is greater or equal to 1 compared to placebo-treated offspring.
Genome_build: mm10
Supplementary_files_format_and_content: TXT, raw non-normalized counts

source_name: liver

growth_protocol: Newborn pups were monitored for survival every 8 hours for a total of 24 hours. Newborn pups that died within 24 hours were harvested immediately for liver and lung tissues. Surviving newborn pups were euthanized by decapitation with scissors. The lung and liver tissues were preserved in RNA later (Qiagen, Valencia, CA) for RNA extraction.

extract_protocol:
Total RNA in the lung and liver tissue was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) from 3 independent experiments per for alive Wild-type, alive Man1a2 heterozygous and dead Man1a2 null, and 2 independent experiments for dead Man1a2 heterozygous pups.
Purified RNA was subjected to library synthesis using Illumina Truseq stranded mRNA kit. Purified RNA was fragmented using divalent cations under elevated temperature, and fragments copied into first stand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. Aftering adding a single 'A' base, cDNA was ligated to the adapter, purified and enriched with PCR to create the final cDNA library. Libraries were validated using KAPA Biosystems primer premix kit with Illumina-compatible DNA primers and Qubit 2.0 fluorometer. Quality of libraries was examined using Agilent Bioanalyzer Tapestation 2200 and final libraries were pooled at a final concentration of 1.8 pM. Cluster generation and 150bp paired read dual-indexed sequencing was performed on Illumina Novaseq to obtain 20 million reads.

treatment_protocol: Man1a2 heterozygous male and female mice were fed with NAC solution (1g/kg body weight) or with placebo (water) one week prior to breeding and continued until delivery of offspring.

tag: strain background

value: Man1a2 null mutant

value: placebo

value: liver


title: hda1-3_NETseq

tag: strain

tag: genotype/variation


title: hda1-2_NETseq


title: X51_LCP0024_B


title: X32_LCP0017_B


title: NGP_control_1

tag: genotype

value: Neuroblastoma NGP cells


title: Man1a2 heterozygous dead placebo liver [PHD_Liver_SIN238A11_S22]

value: Man1a2 Het mutant


title: hda1-4_NETseq


title: X48_LCP0023_B


title: hda1-1_NETseq


title: DNAseq of Arabidopsis: Col-0_rep2


title: gcn5-4_NETseq

value: RPB3::RPB3-3xFLAG NAT, GCN5::HIS3


title: Naïve-mouse-1


title: X54_LCP0024_B


title: Man1a2 heterozygous alive placebo liver [PHA_Liver_SIN238A10_S13]


title: Man1a2 heterozygous alive NAC liver [NHA_Liver_SIN238A14_S49]

value: N-acetyl cysteine (NAC)


title: X29_LCP0016_B


title: Man1a2 homozygous dead NAC liver [NND_Liver_SIN238A13_S40]


title: DNAseq of Arabidopsis: ddm1-1_nrpd1_rep1


title: DNAseq of Arabidopsis: Col-0_rep1


title: X22_LCP0014_B


title: gcn5-2_NETseq

value: Nascent RNA


title: X17_LCP0012_B


title: pDC-depleted_H781L_M_NS

city: New York

first: Yelena

last: Nemirovskaya

address: 1230 York Avenue

institute: Rockefeller University

description:
pDC-depleted_H781L_M_NS
pDC-depleted_gene_expression.txt

data_processing:
quality check by fastqc
alignment by STAR
read count by FeatureCounts
normalization by DESeq2
Genome_build: hg19
Supplementary_files_format_and_content: text, normalized and log2-transformed read counts

source_name: pDC-depleted

extract_protocol:
RNeasy Micro kits
SMART-Seq® v4 PLUS Kit

treatment_protocol: non-stimulated, CpG-C, SARS-CoV-2

tag: gender

value: female

value: healthy

tag: stimulation

value: NS


title: Man1a2 homozygous dead placebo lung [PND_Lung_SIN238A1_S2]

source_name: lung

value: lung


title: pDC-depleted_H781L_P1_LPS

description:
pDC-depleted_H781L_P1_LPS
pDC-depleted_gene_expression.txt

value: male

value: LPS


title: gcn5-3_NETseq

data_processing:
Library strategy: NET-seq
For NET-seq, libraries were sequenced in 76 base pair reads for all NextSeq libraires. For reads shorter than 75 basepairs respectively the adapter sequenice (ATCTCGTATGCCGTCTTCTGCTTG) was trimmed from the fastq files and files were filtered using PrinSeq version prinseq-lite-0.20.2 (http://prinseq.sourceforge.net). The linker contained a molecular barcode with a random hexamer sequence. These random hexamers were removed using custom python scripts. Data were then aligned to the SacCer3 genome using the TopHat2 aligner v2.0.5 and reverse transcription mispriming events are identified and removed where molecular barcode sequences correspond exactly to the genomic sequence adjacent to the aligned read. Only the position corresponding to the 5’ end of the sequencing read (after removal of the barcode), which corresponds to the 3’ end of the nascent RNA fragment, is recorded with a custom python script using HTSeq package in python.
For ChIP-nexus, all reads were filtered for the presence of the fixed barcode (CTGA), beginning at position 6 of the read. Next, the adapter sequence was trimmed using cutadapt with the following parameters: cutadapt -f fastq -a AGATCGGAAGAGCACACGTCT -O 3 -m 22 --length-tag ‘length=’. Lastly, before alignment to the genome, the molecular barcode was removed using custom python scripts. As the ChIP-nexus samples were spiked with S. pombe, the reads were aligned to a combined genome containing both the S. cerevisiae and S. pombe genomes. Reads were aligned using Bowtie version 1.1.1 (Langmead et al., 2009) with the following parameters: -v 2 -M 1 --best -S. Plus and minus strand coverage were then combined and coverage files were then generated using BEDtools version 2.23.0 with the following parameters: genomeCoverageBed -d -5. Only the 5’ end of the read, which corresponds to the point of crosslinking is recorded. The S. cerevisiae and S. pombe data were then separated and S. cerevisiae coverage files were normalized by the total number of mapped S. pombe reads.
Genome_build: sacCer3
Supplementary_files_format_and_content: Bedgraph with 4 columns: Chromosome, Start, End, Score

extract_protocol:
For NET-seq, frozen cells were cryogenically pulverized for lysis and nascent RNA was isolated via an immunoprecipitation at 4°C of Rpb3-3xFLAG and purified using the miRNeasy kit (Qiagen, 217004). For ChIP-nexus cells were crosslinked in 1% formaldehyde, lysed by bead beating, and sonicated. Chomatin was isolated via immunoprecipitation using antibodies: for H3 (Abcam, ab1791), H4 (Abcam, ab10158), H3K23Ac (Millipore, 07-355), and H4K12Ac (Millipore, 07-595), IPs, 12 µg of each antibody were added to 100 µL of Protein G Dynabeads (Thermo Fischer Scientific) previously washed 3 times with 1 mL of 0.5% BSA in PBS.
For NET-seq, a preadenylated RNA linker was ligated onto the 3’ end of the immunoprecipitated RNA. Fragmentation of the ligated samples by alkaline hydrolysis followed by a size selection after electrophoresis on a polyacrylamide gel allowed for the final DNA library to contain inserts of a narrow range. cDNA synthesis by reverse transcription (SuperScriptsIII (Invitrogen)) was performed with a long 5’ phosohorylated primer split by two 18 carbon spacers. Circularization of the RT product was performed with CircLigase (Epicentre) according to the manufacturer’s directions. The PCR was performed directly on the circularized product resulting in DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site positioned so that sequencing would start at the 3’ end. DNA was purified from a PCR reaction that had not reached saturation (~8-10 cycles) by band extraction from a polyacrylamide gel. DNA was sequenced on the Illumina NextSeq 500 according to the manufacturer’s instructions in 75 base pair reads. Preparation of ChIP-nexus libraries was done as described in (He et al., 2015) with the following modifications made to adapt the protocol to yeast: Frozen cell pellets were thawed on ice 1600µL of LB 140 (50 mM HEPES pH 7.4, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1X protease inhibitors (Roche, cOmplete EDTA free)) was added to the pellet. The samples were then split into two screw-cap tubes and acid-washed glass beads (Sigma) were added to the meniscus. Cells were lysed by bead beating for a total of 8 minutes, beating for 1 minute at 4˚C followed by resting for 2 minutes on ice. Chromatin was then removed from the tubes by using a 25G needle to pierce the bottom of the tube. The tubes were then placed inside a 15 mL conical and spun for 1 minute at 1000 xg at 4˚C. The samples were then transferred to a 1.5 mL eppendorf tube and spun at 14,000 rpm for 5 minutes at 4˚C. Pellets were next washed in 800 µL of LB 140 and collected again by centrifuging at 14,000 rpm for 5 minutes at 4˚C. 800 µL of LB 140 was added to the sample and the suspension was transferred to a 15 mL polystyrene tube. 600 µL of LB 140 was used to wash the empty eppendorf tube and then added to the 15 mL tube. Chromatin was then sonicated using Diagenode Bioruptor. Six cycles of sonication were done, each for 5 minutes and 30 seconds ON and 30 seconds OFF for a total sonication time of 15 minutes. After sonication, the chromatin was spun at 20,000 xg for 20 minutes at 4˚C. The supernatant was transferred to a new tube and the concentration of the chromatin was assayed by a Modified Lowry assay (Pierce). The size of the sonicated chromatin was confirmed on a 1.5% agarose gel to be between 100 - 400 bp. For each IP, 3 mg of chromatin extract was used, spiked with 5% (by protein concentration) of S. pombe chromatin. Samples were diluted up to 10 mL using WB 140 (50mM HEPES pH 7.4, 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate). For H3 (Abcam, ab1791), H4 (Abcam, ab10158), H3K23Ac (Millipore, 07-355), and H4K12Ac (Millipore, 07-595), IPs, 12 µg of each antibody were added to 100 µL of Protein G Dynabeads (Thermo Fischer Scientific) previously washed 3 times with 1 mL of 0.5% BSA in PBS. After the final was, the supernatant was removed and the chromatin was added to the beads in a 15 mL conical tube. Samples were incubated overnight at 4˚C with rotation. The remainder of the library construction was done exactly as described in (He et al., 2015). The only modifications were changes made to the four wash buffers to adapt them to yeast. Wash buffer A consisted of 10mM TE and 0.1% Triton X-100. Wash buffer B consisted of 50mM HEPES pH 7.4, 140mM NaCl, 1mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate. Wash buffer C is the same as wash buffer B, but with 250mM NaCl. Wash buffer D consisted of 10mM Tris pH 7.5, 250 mM lithium chloride, 10 mM EDTA, 0.5% NP40, 0.1% sodium deoxycholate.

value: IP against 3xFLAG affinity label


title: elf1-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, ELF1::HIS3


title: pDC-depleted_H781L_P1_COV2

description:
pDC-depleted_H781L_P1_COV2
pDC-depleted_gene_expression.txt


title: X19_LCP0013_B

value: Non-tumor


title: elf1-2_NETseq


title: Man1a2 homozygous dead NAC lung [NND_Lung_SIN238A5_S38]

value: Man1a2 null mutant


title: pDC-depleted_H781L_P1_NS

description:
pDC-depleted_H781L_P1_NS
pDC-depleted_gene_expression.txt


title: pDC-depleted_H781L_P1_CpG

description:
pDC-depleted_H781L_P1_CpG
pDC-depleted_gene_expression.txt

value: CpG


title: gcn5-1_NETseq

description: nascent RNA isolated by IP against Rpb3-3x FLAG affinity label


title: X5_LCP0007_B


title: Wild-type NAC liver [NWA_Liver_SIN238A16_S67]

value: Wild-type

value: liver


title: X35_LCP0018_B


title: eaf1-4_NETseq

value: RPB3::RPB3-3xFLAG NAT, EAF1::HIS3


title: Man1a2 heterozygous dead placebo lung [PHD_Lung_SIN238A3_S20]

value: Man1a2 Het mutant

value: placebo


title: eaf1-2_NETseq

value: Nascent RNA


title: Wild-type placebo liver [PWA_Liver_SIN238A12_S31]


title: X10_LCP0009_B


title: eaf1-1_NETseq


title: Man1a2 heterozygous alive NAC lung [NHA_Lung_SIN238A6_S47]

value: N-acetyl cysteine (NAC)


title: Wild-type placebo lung [PWA_Lung_SIN238A4_S29]


title: Man1a2 heterozygous dead NAC lung [NHD_Lung_SIN238A7_S56]


title: pDC-depleted_H781L_M_LPS

description:
pDC-depleted_H781L_M_LPS
pDC-depleted_gene_expression.txt

value: female

value: healthy


title: X26_LCP0015_B


title: pDC-depleted_H781L_M_CpG

description:
pDC-depleted_H781L_M_CpG
pDC-depleted_gene_expression.txt


title: pDC-depleted_H630Y_P1_LPS

description:
pDC-depleted_H630Y_P1_LPS
pDC-depleted_gene_expression.txt


title: pDC-depleted_H630Y_P1_CpG

description:
pDC-depleted_H630Y_P1_CpG
pDC-depleted_gene_expression.txt


title: dhh1-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, DHH1::HIS3


title: pDC-depleted_H781L_M_COV2

description:
pDC-depleted_H781L_M_COV2
pDC-depleted_gene_expression.txt


title: Wild-type NAC lung [NWA_Lung_SIN238A8_S65]

value: lung


title: Man1a2 heterozygous alive placebo lung [PHA_Lung_SIN238A2_S11]


title: dst1-1_NETseq

value: RPB3::RPB3-3xFLAG NAT, DST1::HIS3


title: ctr9-4_NETseq

value: RPB3::RPB3-3xFLAG NAT, CTR9::HIS3

value: IP against 3xFLAG affinity label


title: X1_LCP0006_B

value: Non-tumor


title: NPC repl3

city: Zurich

first: Raffaella

last: Santoro

address: Winterthurerstrasse 190

institute: University of Zurich

data_processing:
Fastaq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
The bam files were analyzed using the damidseq pipeline script from the Brand group (http://owenjm.github.io/damidseq_pipeline) (Marshall and Brand, 2015). The pipeline bins the mapped reads into GATC-fragments according to GATC-sites indicated by a gff file for the GRCm38 mouse genome (already provided by the authors of the pipeline on the website above) and normalizes reads against the Dam control, in our case the H2B-Dam sample. The pipeline gives a bedgraph file with the log2 ratio of the m6A between H2B-Dam-NoLS and the H2B-Dam only.  The bedgraph files were processed with the find_peaks software associated with the pipeline (https://github.com/owenjm/find_peaks) adjusting the values of the FDR and the minimum quantile (FDR <0.01 and min_quant 0.70). Only the significative peaks common to both replicates were considered as NADs for further analysis. The intersection of identified NADs and previously published lamina associated domains (LADs, Peric-Hupkes et al., 2010) results in the classification of NADonly, NAD/LAD, and LAD.
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
for RNA SEQ, reads were aligned to the reference genome (ensembl version 82) with Subread (i.e. subjunc, version 1.4.6-p4; 50) allowing up to 16 alignments per read (options: –trim5 10 –trim3 15 -n 20 -m 5 -B 16 -H –allJunctions). Count tables were generated with Rcount 51 with an allocation distance of 100 bp for calculating the weights of the reads with multiple alignments, considering the strand information, and a minimal number of 5 hits. For each gene, exon coverage was calculated using a custom pipeline and then normalized in reads per kilobase per million (RPKM) (Mortazavi et al., 2008), the method of quantifying gene expression from RNA sequencing data by normalizing for total read length and the number of sequencing reads.
Genome_build: mm10

source_name: Neural progenitors cells

growth_protocol: Neural progenitor cells were generated from ESCs, according to a previously established protocol (Bibel et al., 2004) using a suspension-based embryoid bodies formation (Bacteriological Petri Dishes, Bio-one with vents, Greiner). The neural differentiation media (DMEM, 10% fetal calf serum, 1× MEM NEAA, Pen/Strep, β-mercaptoethanol, Sodium Pyruvate) was filtered through 0.22 μm filters and stored at 4°C. During the 8-day differentiation procedure, media was exchanged every 2 days. In the last 4 days of differentiation, the media was supplemented with 2 μM retinoic acid to generate neural precursors that are Pax-6-positive radial glial cells.

extract_protocol:
Total RNA from three independent mouse NPC-derived colture dishes was purified with TRIzol reagent (Life Technologies). In order to remove DNA contaminants, the samples were treated with 1U DNaseI (Thermo Scientific) for 1h at 37°C and the RNA samples were re-purified using TRIzol. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany).
The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit HS4000 (Illumina, Inc, California, USA) was used for cluster generation using 10 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the NovaSeq 6000 single end 100 bp using the TruSeq SBS Kit HS4000 (Illumina, Inc, California, USA).

tag: cell line

value: NPC derived in vitro from E14

tag: antibody


title: eaf1-3_NETseq


title: X67_LCP0032_B


title: X65_LCP0031_B


title: X7_LCP0008_B


title: NPC repl1


title: pDC-depleted_H630Y_P1_COV2

description:
pDC-depleted_H630Y_P1_COV2
pDC-depleted_gene_expression.txt

value: male


title: NPC repl2


title: pDC-depleted_H630Y_Br2_NS

description:
pDC-depleted_H630Y_Br2_NS
pDC-depleted_gene_expression.txt

value: NS


title: X13_LCP0010_B


title: ctr9-3_NETseq


title: X45_LCP0021_AR

value: Margin


title: X42_LCP0020_AR


title: pDC-depleted_H630Y_Br2_CpG

description:
pDC-depleted_H630Y_Br2_CpG
pDC-depleted_gene_expression.txt

value: CpG