title: K562_ONT_PlaB_1

description:
4sU labeled chromatin-associated RNA
K562 PlaB 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1

source_name: K562 cells

growth_protocol: K562 cells (ATCC, CCL-243) were maintained at 37°C and 5% CO2 in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin.

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (Clontech/Takara, 2180), incubating for 7.5 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.


title: K562_4sUchr_ONT_noA

status: Public on Dec 12 2019

type: SRA

city: Sherbrooke

first: Karine

last: Choquet

state: Québec

address: 3201 rue Jean-Mignault

department: Biochemistry and Functional Genomics

country: Canada

institute: Université de Sherbrooke

description:
K562 4sU labeled chromatin-associated RNA with rRNA depletion.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION

library_source: transcriptomic

data_processing:
For Illumina RNA-seq datasets, paired-end reads were aligned to reference genomes using STAR (v2.5.1a) with default parameters (except outFilterMultimapNmax=101, outSJfilterOverhangMin=3 1 1 1, outSJfilterDistToOtherSJmin=0 0 0 0, alignIntronMin=11, alignEndsType=EndToEnd). The splicing index counts file was generated for each gene by counting the number of read pairs that span splice junctions by at least 3 nucleotides. '5SS_count' column represents read pair counts over an intron's 5'SS; '3SS_count' column represents read pair counts over an intron's 3'SS; 'splice_count' represents read pair counts that span the exon-exon junction of a spliced intron.
For Oxford Nanopore Technologies direct RNA sequencing datasets, raw signal fast5 files were basecalled using Albacore 2.2.7 (Oxford Nanopore Technologies Ltd.) with the following parameters: read_fast5_basecaller.py --flowcell FLO-MIN106 --kit SQK-RNA001 --recursive --output_format fast5,fastq --worker_threads 8 --save_path ${savePath} --input ${inputPath}. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) and GMAP (version 2018-03-25) with default parameters. All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
For Oxford Nanopore Technologies direct RNA sequencing, the read ends dataset was generated by intersecting the start position of the read (end position of the RNA) to annotated gene features. “Intron” and “Exon” regions refer to their annotated features within protein coding genes. “Poly(A)” sites are defined as regions within 50 nucleotides of the end coordinate of annotated genes. In K562 datasets, the poly(A) region also includes regions that are within 50 nucleotides of RNA-PET annotations from cytoplasm and chromatin fractions in K562 ENCODE data (40). “Post_poly(A)” sites are defined as the region 50-550 nucleotides after the end of annotated genes. “Splice_sites” are defined as 50 nucleotides upstream and 10 nucleotides downstream of annotated 5’ splice sites. “Undetermined” categorizes reads that do not fit into one category and “other” represents read ends that do not align in the sense direction to annotated gene features (e.g. antisense transcripts, noncoding RNAs, intergenic transcription, etc.).
For Oxford Nanopore Technologies direct RNA sequencing, the splice dataset was generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene).
For Oxford Nanopore Technologies direct RNA sequencing, the intron pairs dataset was generated by identifying reads that span two or more introns. The coordinates and splicing status (from the splice dataset) of the two introns is recorded in the file.
Genome_build: Reference genomes for human K562 and Drosophila S2 sequence alignments were obtained from ENSEMBLE GRCh38 (release-86) and FlyBase dm6 (r6.19), respectively.
Supplementary_files_format_and_content: Tab-delimited ‘*SI_counts.txt’ files contain read pair counts spanning intron junctions. Tab-delimited '*end_stats.txt' files contain the read name and feature that the start of the read (end of the RNA) maps to. Tab-delimited ‘*medIntrons_discarded_splice_df.txt’ files contain the read name, coordinates of the intron, read length, alignment error rate, read start clipped length, distance from the read start (or RNA end) to the 3’SS of the intron, the determined splicing status, gene and poly(A) site separated by ‘_’, and a column for if the RNA ends near or after the gene’s poly(A) site. Tab-delimited ‘*intron_pairs_df.txt’ files contain the read name, intron coordinates, and splicing status of two introns that one read maps to.

molecule: total RNA

organism: Homo sapiens

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using Ribo-Zero Gold rRNA Removal kit (Illumina, MRZG126).
Direct RNA sequencing protocol using the SQK-RNA001 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.

treatment_protocol: Cells were labeled in media containing 500 uM 4-thiouridine for 8 minutes.

tag: cell line

tag: cell treatment

value: 4-thiouridine (4sU), 500 uM, 8 minutes

tag: fractionation

value: chromatin purification

tag: 4su selection

value: 4sU biotinylation and streptavidin pulldown

tag: library prep

value: Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA001 kit


title: K562_4sUchr_ONT_3

description:
K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 3.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using Ribo-Zero Gold rRNA Removal kit (Illumina, MRZG126). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (New England Biolabs, M0276S), incubating for 1 hour at 37°C.
Direct RNA sequencing protocol using the SQK-RNA001 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.


title: K562_4sUchr_2

description:
K562 4sU labeled chromatin-associated RNA biological rep 2.
RNA-seq from cDNA.
4sU labeled chromatin-associated RNA.

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254).
Libraries were prepared using the Ovation Universal RNA-seq System (NUGEN, 0343-32) with Universal Human rRNA strand selection reagent (NUGEN, S01859) following the manufacturer’s instructions.

value: Nugen Ovation Universal RNA-seq System with human rRNA strand selection reagent


title: cmc_wt_74

status: Public on Dec 15 2019

city: Ramat Gan

first: Tirza

middle: T

last: Doniger

state: Not US or Canada

address: Bar Ilan University

department: The Mina and Everard Faculty of Life Sciences

country: Israel

institute: Bar Ilan University

description: control wt 74

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using was aligned using STAR (v 2.5.0a)  (Dobin et al., 2013) with default parameters.
The UCSC mm10 iGenome build and its corresponding gtf were used to build the STAR reference database.
The number of reads per gene were quantified using HTSeq-Count (v0.6.1p1) (Anders et al., 2015).
The R Bioconductor package, DESeq2 (v1.18.1)  (Love et al.,2014),  was used to classify genes as differentially expressed (Benjamini-Hochberg adjusted p-value < 0.1).
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include raw counts

organism: Mus musculus

source_name: liver

extract_protocol:
Livers were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Mice were orally gavaged at 50mg/kg with WY in CMC 0.5% or CMC 0.5% and sacrificed 12hours later.

tag: strain

tag: tissue

value: liver

tag: genotype

value: wild type

tag: treatment

value: carboxymethyllcellulose control

tag: age

value: 11-13 months


title: K562_4sUchr_1

description:
K562 4sU labeled chromatin-associated RNA biological rep 1.
RNA-seq from cDNA.
4sU labeled chromatin-associated RNA.


title: K562_4sU_2

description:
K562 4sU labeled RNA biological rep 2.
RNA-seq from cDNA.
4sU labeled RNA.

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL. Cells were collected by centrifugation at 500 g for 2 minutes and washed once in 1X PBS. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. 4sU labeled RNA was purified as previously described in (Dolken, NRA, 2008) and (Schwalb, Science, 2016). Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254).
Libraries were prepared using the Ovation Universal RNA-seq System (NUGEN, 0343-32) with Universal Human rRNA strand selection reagent (NUGEN, S01859) following the manufacturer’s instructions.


title: K562_4sU_1

description:
K562 4sU labeled RNA biological rep 1.
RNA-seq from cDNA.
4sU labeled RNA.


title: K562_chr_1

description:
K562 chromatin-associated RNA biological rep 1.
RNA-seq from cDNA.
Chromatin-associated RNA.

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions.
Libraries were prepared using the Ovation Universal RNA-seq System (NUGEN, 0343-32) with Universal Human rRNA strand selection reagent (NUGEN, S01859) following the manufacturer’s instructions.


title: K562_tot_2

description:
K562 total RNA biological rep 2.
RNA-seq from cDNA.

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and collected by centrifugation at 500 g for 2 minutes. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions.
Libraries were prepared using the Ovation Universal RNA-seq System (NUGEN, 0343-32) with Universal Human rRNA strand selection reagent (NUGEN, S01859) following the manufacturer’s instructions.


title: S2_4sUchr_ONT_1a

description:
S2 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1 technical rep a.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION

organism: Drosophila melanogaster

source_name: S2 cells

growth_protocol: S2 cells (Expression Systems, 94-005) were maintained at 25°C in Schneider’s Drosophila medium containing 10% heat inactivated FBS, 50 U/ml penicillin and 50 ug/ml streptomycin.

extract_protocol:
S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded immediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylalcohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using Ribo-Zero Gold rRNA Removal kit (Illumina, MRZG126). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (New England Biolabs, M0276S), incubating for 1 hour at 37°C.
Direct RNA sequencing protocol using the SQK-RNA001 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.


title: BL1184_4sUchr_ONT_1

description: BL1184 4sU labeled chromatin-associated RNA with rRNA depletion and poly(I) tail addition biological rep 1

data_processing:
For Illumina RNA-seq datasets, paired-end reads were aligned to reference genomes using STAR (v2.5.1a) with default parameters (except outFilterMultimapNmax=101, outSJfilterOverhangMin=3  1  1  1, outSJfilterDistToOtherSJmin=0  0   0   0, alignIntronMin=11, alignEndsType=EndToEnd). The splicing index counts file was generated for each gene by counting the number of read pairs that span splice junctions by at least 3 nucleotides. '5SS_count' column represents read pair counts over an intron's 5'SS; '3SS_count' column represents read pair counts over an intron's 3'SS; 'splice_count' represents read pair counts that span the exon-exon junction of a spliced intron.
For Oxford Nanopore Technologies direct RNA sequencing datasets, raw signal fast5 files were basecalled using Albacore 2.2.7 (Oxford Nanopore Technologies Ltd.) with the following parameters: read_fast5_basecaller.py --flowcell FLO-MIN106 --kit SQK-RNA001 --recursive --output_format fast5,fastq --worker_threads 8 --save_path ${savePath} --input ${inputPath}. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) and GMAP (version 2018-03-25) with default parameters. All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
For Oxford Nanopore Technologies direct RNA sequencing, the read ends dataset was generated by intersectin the start position of the read (end position of the RNA) to annotated gene features. “Intron” and “Exon” regions refer to their annotated features within protein coding genes. “Poly(A)” sites are defined as regions within 50 nucleotides of the end coordinate of annotated genes. In K562 datasets, the poly(A) region also includes regions that are within 50 nucleotides of RNA-PET annotations from cytoplasm and chromatin fractions in K562 ENCODE data (40). “Post_poly(A)” sites are defined as the region 50-550 nucleotides after the end of annotated genes. “Splice_sites” are defined as 50 nucleotides upstream and 10 nucleotides downstream of annotated 5’ splice sites. “Undetermined” categorizes reads that do not fit into one category and “other” represents read ends that do not align in the sense direction to annotated gene features (e.g. antisense transcripts, noncoding RNAs, intergenic transcription, etc.).
For Oxford Nanopore Technologies direct RNA sequencing, the splice dataset was generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene)
For Oxford Nanopore Technologies direct RNA sequencing, the intron pairs dataset was generated by identifying reads that span two or more introns. The coordinates and splicing status (from the splice dataset) of the two introns is recorded in the file.
Genome_build: Reference genomes for human K562 and Drosophila S2 sequence alignments were obtained from ENSEMBLE GRCh38 (release-86) and FlyBase dm6 (r6.19), respectively.
Supplementary_files_format_and_content: Tab delimited ‘*SI_counts.txt’ files contain contain read pair counts spanning intron junctions. Tab delimited '*end_stats.txt' files contain the read name and feature that the start of the read (end of the RNA) maps to. Tab delimited ‘*medIntrons_discarded_splice_df.txt’ files contain the read name, coordinates of the intron, read length, alignment error rate, read start clipped length, distance from the read start (or RNA end) to the 3’SS of the intron, the determined splicing status, gene and poly(A) site separated by ‘_’, and a column for if the RNA ends near or after the gene’s poly(A) site. Tab delimited ‘*intron_pairs_df.txt’ files contain the read name, intron coordinates, and splicing status of two introns that one read maps to.

source_name: BL1184 cells

growth_protocol: BL1184 cells (ATCC, CRL-4959) were maintained at 37°C and 5% CO2 in RPMI 1640 medium containing, 2 mM L-Glutamine, 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin.

extract_protocol:
BL1184 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million BL1184 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(I) tail was added to the 3’ ends of the RNA sample with yeast poly(A) polymerase (ThermoFisher, 74225Z25KU) and ITP, incubating for 30 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software on the MinION instrument for 48 hours with default settings for direct RNA sequencing.

value: Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA002 kit


title: BL1184_4sUchr_ONT_2


title: K562_4sUchr_ONT_5b

description: K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(I) tail addition biological rep 5b

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(I) tail was added to the 3’ ends of the RNA sample with yeast poly(A) polymerase (ThermoFisher, 74225Z25KU) and ITP, incubating for 30 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software on the MinION instrument for 48 hours with default settings for direct RNA sequencing.


title: S2_ONT_PlaB_2

description:
4sU labeled chromatin-associated RNA
S2 PlaB 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1b

extract_protocol:
S2 cells in an adherent layer at 95% confluency were lifted with gentle pipetting and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 50 million S2 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(A) tail was added to the 3’ ends of the RNA sample with E. coli poly(A) polymerase (Clontech/Takara, 2180), incubating for 7.5 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software for 48 hours with default settings for direct RNA sequencing.

value: 100 nM PlaB 1 hour; 4-thiouridine (4sU), 500 uM, 8 minutes


title: K562_ONT_PlaB_2

description:
4sU labeled chromatin-associated RNA
K562 PlaB 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2


title: 1 DPA, no pollination (S+1)_F7

status: Public on Nov 03 2019

description:
F7_GAATTCGT-TAATCTTA_L003
S+1_F7

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using FASTX-Toolkit 0.0.13
Reads were aligned to the Cucumber Chinese Long genome (v2) using STAR (v2.5.0a)
HTSeq v0.6.1p1 was used to count the number of reads mapped to each gene
Genome_build: C. sativus L. var. sativus cv. 9930 (Chinese Long v2) at:
ftp://cucurbitgenomics.org/pub/cucurbit/genome/cucumber/Chinese_long/v2/
Supplementary_files_format_and_content: tab-delimited text files include raw counts and DESEQ2 normalized values for each gene in each Sample

organism: Cucumis sativus var. sativus

source_name: 1 DPA, no pollination (S+1)_ovary

growth_protocol: Net-house experiment with mature plants of gynoecious non-parthenocarpic Line Elem Female.

extract_protocol:
RNA was prepared from 50 mg of frozen cucumber ovary tissue using the RiboEx (GeneAll, cat. no 301-902) Tri-Reagent protocol (Sigma-Aldrich, USA). First-strand cDNA was synthesized with a Random primer using qScript Flex cDNA Kit (Quanta).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: pollination with pollinator genotype and sampling performed around 7 am

tag: genotype/variation

tag: developmental stage

value: 1 DPA, no pollination (S+1)

value: Ovary


title: 1 day after after pollination in presence of older fruits (f+1)_B8

description:
B8_CTGAAGCT-TAATCTTA_L003
f+1_B8

source_name: 1 day after after pollination in presence of older fruits (f+1)_ovary

value: gynoecious non-parthenocarpic Line Elem Female


title: K562_4sUchr_ONT_1

description:
K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION


title: K562_ONT_DMSO_2

description:
4sU labeled chromatin-associated RNA
K562 DMSO 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2

value: 0.01% DMSO 1 hour; 4-thiouridine (4sU), 500 uM, 8 minutes


title: S2_ONT_PlaB_3

description:
4sU labeled chromatin-associated RNA
S2 PlaB 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2


title: K562_ONT_DMSO_1

description:
4sU labeled chromatin-associated RNA
K562 DMSO 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 1


title: K562_4sUchr_ONT_2

description:
K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2.
Direct RNA nanopore sequencing.
4sU labeled chromatin-associated RNA.
Oxford Nanopore Technologies MinION


title: mouse liver-308-HFD,RNA-s102796-001-038_HFD.fastq.R1

status: Public on Sep 04 2019

city: Leiden

first: Martine

middle: C

last: Morrison

address: Sylviusweg 71

department: MHR

country: Netherlands

institute: TNO

data_processing:
Clustering and DNA sequencing were performed by GenomeScan BV (Leiden, Netherlands) using a NextSeq 500 sequencer (Illumina, San Diego, California, USA) with a single-read 75-cycle sequencing protocol with at least 15 million reads per sample and %Q30 > 92.58-93.75%.
Fastq files of the same sample (from mutiple sequence runs an/or lanes) were merged to 1 fastq-file/sample.
Reads of the fastq files were aligned to the mouse reference genome (GRCm38p4) using the Star2.5 alignment algorithm. The default mismatch rate of 2% was used (3 mismatches in a read of 150 bases). The alignment files (BAM files) were sorted and indexed with the Samtools v0.1.18 package.
Htseq-count 0.6.1p1 was used to count the read mapping frequency/gene (transcript region) resulting in count.files.
Genome_build: Genome Reference Consortium Human Build 38 patch release 7 (GRCh38.p7)
Supplementary_files_format_and_content: The count files contain the # reads mapped per gene (not normalised per sample), the bottom lines contain a summary of (un-)mapped reads.

source_name: mouse liver

growth_protocol: Animals and Diets   All animal experiments were performed according to the rules and regulations set out by the Netherlands Law on Animal Experiments and were approved by an independent Committee on Animal Care and Experimentation (approval reference number DEC-3383, Date: 25 February 2013, DEC-Zeist, The Netherlands). Male Ldlr−/−.Leiden mice [17,18] were bred by TNO Metabolic Health Research (Leiden, The Netherlands). Mice were housed in Makrolon® cages at approximately 21 °C, with a 12-h daily light cycle and relative humidity of 50–60%. The mice were supplied with food and tap water ad libitum and mice were fed standard lab chow (Ssniff R/M diet V1530, Uden, The Netherlands) until they were 12–15 weeks old.

extract_protocol:
Food Intake, Body Composition    Food intake and body weight were monitored every three weeks of the study. Total body fat mass was assessed with EchoMRI at the start and in week 9, 15 and 21 of the study as previously described [42].   Plasma Measurements   Blood samples were collected after 5 h fast via tail vein bleeding for isolation of EDTA plasma for evaluation of total cholesterol, triglycerides and insulin as described [17]. Plasma HDL-cholesterol was determined after precipitation as described earlier [43] with enzymatic assays (CHOD-PAP, Roche Diagnostics, Almere, the Netherlands). Non-HDL cholesterol was calculated by subtracting HDL-cholesterol levels from total plasma cholesterol levels. Blood glucose was measured using a hand-held glucometer in tail blood during blood collection (FreeStyle Lite, Abbott, Alameda CA, USA).    Sacrifice and Analysis of Fat and Liver Tissue   All animals were sacrificed under non-fasted conditions by CO2 asphyxiation after 21 weeks of dietary treatment. Blood was collected via cardiac puncture for EDTA-plasma collection. White adipose tissue (WAT) was collected from gonadal and mesenteric WAT, abdominal fat mass, and the subcutaneous fat depot. The liver was weighed and divided into different parts. The medial lobe was fixed in formalin and embedded in paraffin. Development of NAFLD was analyzed in 3-μm liver hematoxylin-eosin stained sections and analyzed by a board-certified pathologist using a wellestablished adapted scoring method for human NAFLD [44] as described [17]. Briefly, the level of total steatosis per mouse was expressed as a percentage of the total liver section affected by microvesicular or macrovesicular steatosis. Similarly, hypertrophy was expressed as the affected percentage of the total liver section. Steatosis and hypertrophy were scored in the liver sections at a 50x magnification. The HE stained liver sections were digitalized with a 20 × magnification (Aperio AT2, Leica Biosystems, Amsterdam, The Netherlands) and used for the representative images.   4-HNE Oxidative Stress-Related Marker Staining and Quantification in Liver Tissue    Oxidative stress-related marker 4-hydroxynonenal (4-HNE) was analyzed in 3-μm paraffin liver sections. After deparaffinization of the liver sections, heat-induced epitope retrieval was performed with a PH 6 citrate buffer (PT link, Dako, Denmark). Subsequently, sections were washed with 0.05% Tween-20 in phosphate-buffered saline (PBS) and blocked for 30 min at room temperature (RT) with 5% normal goat serum in PBS. Staining was performed by overnight incubation at 4 °C with Rabbit anti-4-HNE Michael adducts primary antibody (1:1000 in PBS, ref.393207, Millipore Corporation, Billerica, MA, USA PBS). The next day these sections were washed and blocked for endogenous peroxidase with 3% H2O2 during 20 min, and subsequently incubated for 45 min with a BrightVision poly-HRP-anti-rabbit secondary antibody (1:1 in PBS, Immunologic, Duiven, The Netherlands;) all at RT. Lastly, after washing the sections, Diaminobenzidine (DAB) was used for color development by 5 min incubation and nuclei were stained with hematoxylin for 30 s at RT. The 4-HNE stained liver sections were digitalized with a 20 × magnification (Aperio AT2, Leica Biosystems, Amsterdam, The Netherlands). In these scans 4-HNE positive immunoreactivity was scored in two different liver sections in five nonoverlapping fields per liver section and expressed as amount of 4-HNE positive immunoreactivity structures per mm2.    LC-MS/MS   Sacrifice plasma samples were used to measure L-carnitine, nicotinamide and several acylcarnitine levels. The metabolites were measured with the Metabolon analytical system (Metabolon, Inc., Durham, NC, USA). A non-targeted semiquantitative liquid chromatography– tandem mass spectrometry (LC-MS/MS) platform was applied for the identification of structurally named and unknown molecules [45,46]. All normalized relative ion counts were log transformed, and the remaining data were imputed with the minimum value on a per metabolite basis and reported in relative units (RU).
Gene Expression and Pathway Analysis   Total-RNA isolation kit (Bio-connect, Huissen, The Netherlands) was used to extract RNA from snap-frozen livers. The RNA concentration was measured spectrophotometrically using a NanoDrop 1000 (Isogen Life Science, De Meern, The Netherlands). To assess the quality of the isolated RNA, the 2100 Bioanalyzer (Agilent Technologies, Amstelveen, The Netherlands) was used. Next, messenger RNA (mRNA) library was produced for next generation sequencing following the manufacturer′s protocol NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The quality and yield of the PCR products were consistent with the expected size distribution (300–800 base pairs). Clustering and DNA sequencing were performed by GenomeScan BV (Leiden, The Netherlands) using a NextSeq 500 sequencer (Illumina, San Diego, CA, USA). The sequences were directly aligned with the mouse reference genome (GRCm38p4) using the Start 2.5 algorithm with default settings.
RNA-seq was performed by GenomeScan B.V. (Leiden, NL) bu using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #7420S/L, New England Biolabs, Ipswich, Massachusetts, USA). Briefly, mRNA was isolated from total RNA using oligo-dt magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed which was used for ligation with sequencing adapters, PCR amplification and quality measurement by Fragment-analyser. Size distribution generaly was a broad peak between 300-500bp. Clustering and DNA sequencing was performed on Illumina NextSeq 500 using 1.6 pM DNA.

treatment_protocol: Treatments  Subsequently, 74 mice were divided into five experimental groups. The healthy reference group continued to receive a chow diet (n = 13) and the high-fat diet control group (HFD) received a NAFLD inducing diet (45 kcal% fat from lard, 35 kcal% from carbohydrates, primarily sucrose and 20% kcal from protein; D12451, Research Diets, New Brunswick, NJ, USA) as described previously [17] (n = 16). L-carnitine (0.4% w/w, Sigma-Aldrich, Zwijndrecht, The Netherlands; LC group; n = 15), nicotinamide riboside (0.3% w/w, NovAlix, Cedex, France; NR group; n = 15) and the combination of both (COMBI group, n = 15) were mixed through the HFD and the respective groups were treated with these diets for 21 weeks.   Subsequently, 74 mice were divided into five experimental groups. The healthy reference group continued to receive a chow diet (n = 13) and the high-fat diet control group (HFD) received a NAFLD inducing diet (45 kcal% fat from lard, 35 kcal% from carbohydrates, primarily sucrose and 20% kcal from protein; D12451, Research Diets, New Brunswick, NJ, USA) as described previously [17] (n = 16). L-carnitine (0.4% w/w, Sigma-Aldrich, Zwijndrecht, The Netherlands; LC group; n = 15), nicotinamide riboside (0.3% w/w, NovAlix, Cedex, France; NR group; n = 15) and the combination of both (COMBI group, n = 15) were mixed through the HFD and the respective groups were treated with these diets for 21 weeks.

tag: sampling time after initiation intervention=treatment=diet

tag: diet (+/-compound)

value: HFD


title: HH18 embryo_scRNA

status: Public on Nov 27 2019

city: Oxford

first: Tatjana

last: Sauka-Spengler

state: Oxfordshire

address: Radcliffe Department of Medicine

department: University of Oxford

country: United Kingdom

institute: MRC Weatherall Institute of Molecular Medicine

description: Wild-type

data_processing:
I1 read: contains the sample index.
R1 read: contains the cell barcode (first 16 nt) and UMI (next 10 nt).
R2 read: contains the RNA sequence.
Sequenced files were demultiplexed and features counted using CellRanger (v2.2.0)
Seurat (v2.3.4) R package was used for data processing
Genome_build: galGal5
Supplementary_files_format_and_content: barcodes.tsv, genes.tsv, matrix.mtx

organism: Gallus gallus

source_name: HH18 embryo_scRNA

growth_protocol: Fertilised wild-type chicken eggs were obtained from Henry Stewart & Co (Norfolk), staged according to Hamburger and Hamilton (1951) (24). All experiments were performed on chicken embryos younger than 12 days of development, and as such were not regulated by the Animals (Scientific Procedures) Act 1986.

extract_protocol:
Isolated cells were then loaded onto the 10X Chromium machine and libraries prepared as per manufacturer's protocol
As per manufacturer's protocol using the 10X Chromium 3' v2 kit

treatment_protocol: Dissected vagal regions from electroporated embryos were dissociated with dispase (1.5mg/ml in DMEM/10mM Hepes pH 7.5) at 37°C for 15min with intermittent pipetting to achieve a single cell suspension and 0.05% Trypsin at 37°C for a final 3min. The reaction was stopped and cells were resuspended in an excess of Hanks buffer. Cells were centrifuged at 500g for 10min and resuspended in Hanks buffer, passed through a 0.22mm filter and centrifuged at 750g for 10min, pelleted cells were resuspended in 500µl Hanks buffer. Fluorescent positive cells were sorted and collected using BD FACS-Aria Fusion.

value: Wild-type

tag: development

value: Embryo

value: Neural crest (E2+ cells)

value: Dissociation and FACS

value: E2:citrine electroporation HH8/9

tag: amplification

value: 10X Chromium v2

tag: assay

value: scRNA-seq


title: K562_chr_2

description:
K562 chromatin-associated RNA biological rep 2.
RNA-seq from cDNA.
Chromatin-associated RNA.

tag: cell line

value: 4-thiouridine (4sU), 500 uM, 8 minutes

value: chromatin purification

value: Nugen Ovation Universal RNA-seq System with human rRNA strand selection reagent


title: mouse liver-306-HFD,RNA-s102796-001-036_HFD.fastq.R1


title: mouse liver-271-Combi,RNA-s102796-001-034_Combi.fastq.R1

value: HFD+LC+NR = HFD+Combi


title: K562_4sUchr_ONT_4

description: K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(I) tail addition biological rep 4

extract_protocol:
K562 cells were harvested in suspension at 0.8-1 million cells/mL and proceeded imediately to cellular fractionation protocol. Cellular fractionation was performed as described in (Mayer, Nat. Protoc., 2016) and based on (Wuarin & Schibler, MCB, 1994). 10 million K562 cells were collected by centrifugation at 500 g for 2 minutes and washed with 1X PBS. The cell pellet was lysed for 2 min with 200 μl cytoplasmic lysis buffer (0.15% (vol/vol) NP-40, 10 mM Tris-HCl (pH 7.0), and 150 mM NaCl), layered over 500 μl of a sucrose cushion (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (wt/vol) sucrose), and nuclei were collected by centrifugation at 16,000 g for 10 minutes. The nuclei pellet was resuspended in 800 μl wash buffer (0.1% (vol/vol) Triton X-100, 1 mM EDTA, in 1X PBS) and collected by centrifugation at 1,150 g for 1 minute. Washed nuclei were resuspended in 200 μl glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (vol/vol) glycerol, 0.85 mM DTT), and mixed with 200 μl nuclei lysis buffer (1% (vol/vol) NP-40, 20 mM HEPES (pH 7.5), 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT) before pulse vortex and incubation on ice for 2 minutes. The chromatin pellet was collected by centrifugation at 18,500 g for 2 minutes and resuspended in 1X PBS. All steps were performed at 4°C and all buffers were prepared with 25 μM α-amanitin, 0.05U/μl SUPERase.In, and protease inhibitor mix. Samples were immediately resuspended in Qiazol lysis reagent and RNA was extracted following manufacturer's instructions. Labeled RNA (1 μg / 10 μl) was incubated with 10% biotinylation buffer (100mM Tris pH 7.5, 10mM EDTA) and 20% EZ-Link Biotin-HPDP (1mg/mL resuspended in DMF, Thermo Fisher Scientific, 21341) for 1.5 hours at 800 rpm and 24°C in the dark. RNA was purified by mixing with a 1:1 ratio of chloroform/isoamylacohol (24:1), separating with a phase-lock tube at 16000 g for 5 min, and performing isopropanol precipitation. Biotinylated RNA separation was performed using the μMACS streptavidin kit (Miltenyi Biotec, 130-074-101). RNA was mixed with μMACS streptavidin beads at a 2:1 ratio at 800 rpm and 24°C for 15 min. RNA-streptavidin beads mix were transferred to the μMACS column and washed with wash buffer (100mM Tris pH 7.5, 10mM EDTA, 1M NaCl, 0.1% Tween 20) at 65°C and RT each 3 times. Selected RNA was eluted off the magnet with 0.1M DTT and purified using the miRNeasy micro kit (Qiagen, 217084) with on column DNase I treatment (Qiagen, 79254). Ribosomal RNAs were depleted from the 4sU-selected chromatin-associated RNA sample using RiboMinus Eukaryotic Kit v2 (ThermoFisher, A15020). A poly(I) tail was added to the 3’ ends of the RNA sample with yeast poly(A) polymerase (ThermoFisher, 74225Z25KU) and ITP, incubating for 30 min at 37°C.
Direct RNA sequencing protocol using the SQK-RNA002 kit (Oxford Nanopore Technologies Ltd.) was followed exactly as described by the manufacturer with minor modifications described below. 500 ng of RNA sample was ligated to the provided splinted adapter with T4 DNA ligase for 15 minutes at room temperature. Ligated RNA sample was reverse transcribed using SuperScript III (Invitrogen, 18080044), as recommended by Oxford Nanopore Technologies to improve the reading of RNA through the nanopore. Samples were purified with with Agencourt RNAClean XP beads (Beckman Coulter, A63987) and ligated to the sequencing adapter with preloaded motor protein. After a second purification step with Agencourt RNAClean XP beads, the sample was resuspended in Elution Buffer, mixed with RNA Running Buffer, loaded onto a primed FLO-MIN106 flowcell, and sequenced using MINKNOW software on the PromethION instrument for 48 hours with default settings for direct RNA sequencing.

tag: cell treatment

value: 4sU biotinylation and streptavidin pulldown

value: Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA002 kit


title: K562_4sUchr_ONT_5a

description: K562 4sU labeled chromatin-associated RNA with rRNA depletion and poly(I) tail addition biological rep 5a

tag: fractionation


title: 1 DPA, no pollination (S+1)_E7

description:
E7_ATTCAGAA-TAATCTTA_L003
S+1_E7

value: 1 DPA, no pollination (S+1)

value: Ovary


title: mouse liver-212-Chow,RNA-s102796-001-031_Chow.fastq.R1

value: Chow


title: mouse liver-269-Combi,RNA-s102796-001-029_Combi.fastq.R1


title: 1 day after after pollination in presence of older fruits (f+1)_D8

description:
D8_CGGCTATG-TAATCTTA_L003
f+1_D8

value: gynoecious non-parthenocarpic Line Elem Female


title: K562_tot_1

description:
K562 total RNA biological rep 1.
RNA-seq from cDNA.

tag: 4su selection


title: mouse liver-238-NR,RNA-s102796-001-027_NR.fastq.R1

value: HFD+NR


title: 1 DPA, no pollination (S+1)_D7

description:
D7_AGCGATAG-AGGCGAAG_L003
S+1_D7


title: mouse liver-211-Chow,RNA-s102796-001-026_Chow.fastq.R1


title: mouse liver-267-Combi,RNA-s102796-001-024_Combi.fastq.R1


title: 1 day after after pollination (F+1)_H6

description:
H6_TCTCGCGC-AGGCGAAG_L003
F+1_H6

source_name: 1 day after after pollination (F+1)_ovary

value: 1 day after after pollination (F+1)


title: 1 day after after pollination (F+1)_G6

description:
G6_TCCGCGAA-AGGCGAAG_L003
F+1_G6


title: Anthesis (A)_B6

description:
B6_TAATGCGC-AGGCGAAG_L003
A_B6

source_name: Anthesis (A)_ovary

value: anthesis


title: mouse liver-240-NR,RNA-s102796-001-032_NR.fastq.R1


title: Anthesis (A)_H5

description:
H5_GAATTCGT-AGGCGAAG_L002
A_H5


title: Anthesis (A)_A6

description:
A6_CTGAAGCT-AGGCGAAG_L003
A_A6


title: 1 day after after pollination (F+1)_F6

description:
F6_CGGCTATG-AGGCGAAG_L003
F+1_F6


title: mouse liver-252-LC,RNA-s102796-001-023_LC.fastq.R1

value: HFD+LC


title: mouse liver-307-HFD,RNA-s102796-001-037_HFD.fastq.R1

value: HFD


title: mouse liver-237-NR,RNA-s102796-001-022_NR.fastq.R1


title: mouse liver-304-HFD,RNA-s102796-001-030_HFD.fastq.R1


title: mouse liver-258-LC,RNA-s102796-001-033_LC.fastq.R1


title: mouse liver-292-HFD,RNA-s102796-001-020_HFD.fastq.R1


title: mouse liver-272-Combi,RNA-s102796-001-035_Combi.fastq.R1


title: mouse liver-251-LC,RNA-s102796-001-018_LC.fastq.R1


title: S2_ONT_DMSO_2

description:
4sU labeled chromatin-associated RNA
S2 DMSO 4sU labeled chromatin-associated RNA with rRNA depletion and poly(A) tail addition biological rep 2

value: 0.01% DMSO 1 hour; 4-thiouridine (4sU), 500 uM, 8 minutes

tag: library prep


title: mouse liver-256-LC,RNA-s102796-001-028_LC.fastq.R1


title: mouse liver-207-Chow,RNA-s102796-001-016_Chow.fastq.R1


title: mouse liver-264-Combi,RNA-s102796-001-014_Combi.fastq.R1


title: mouse liver-290-HFD,RNA-s102796-001-010_HFD.fastq.R1


title: mouse liver-293-HFD,RNA-s102796-001-025_HFD.fastq.R1


title: mouse liver-265-Combi,RNA-s102796-001-019_Combi.fastq.R1


title: mouse liver-262-Combi,RNA-s102796-001-009_Combi.fastq.R1


title: mouse liver-235-NR,RNA-s102796-001-017_NR.fastq.R1


title: mouse liver-209-Chow,RNA-s102796-001-021_Chow.fastq.R1


title: mouse liver-204-Chow,RNA-s102796-001-006_Chow.fastq.R1


title: mouse liver-246-LC,RNA-s102796-001-008_LC.fastq.R1


title: Piwi CLIP-seq rep2

status: Public on Jul 09 2019

city: Tokyo

first: Yuka

middle: W.

last: Iwasaki

address: 35 Shinanomachi, Shinjuku-ku,

department: Department of Molecular Biology

country: Japan

institute: Keio University School of Medicine

data_processing:
Library strategy: CLIP-Seq
3′ adapter sequences were trimmed using Cutadapt and reads shorter than 27 bp were discarded. We removed PCR duplicates by collapsing all identical reads containing the same random barcode at the 5′ end with fastx-collapser from the FASTX-Toolkit. Also, 5′-end random barcode located at positions 1–9 of the reads were removed. Mapping was then performed against the Release 3 assembly of the Drosophila melanogaster genome with STAR. Alignment output BAM files were sorted and indexed by SAMtools. Peaks were called using Piranha peak-calling algorithm, and reads within the peaks were counted for two replicate samples. Genome-mapped reads were further mapped to TE consensus sequences from Repbase using Bowtie allowing only unique mapping reads. Mapped reads were counted for each consensus TE, and normalized to RPM by the number of Drosophila genome-mapped reads. CLIP tags were compared to SMInput (size-matched input), and enrichment against SMInput was discussed in every analysis.
processed data files format and content: Normalized or raw read counts mapped to each transposon consensus sequence. Only reads mapped to sense direction was counted for CLIP analysis, and only reads mapped to antisense direction was counted for smRNA analysis (Tab delimited format, txt).
Genome_build: dm3

molecule: other

source_name: OSC

growth_protocol: Cells were grown at 26 degrees on OSC medium (Fly Extract added M3 medium)

extract_protocol: Cells were crosslinked, lysed, and limitedly digestested with RNase T1. RNA-protein complexes were immunoprecipitated with indicated antibodies. After dephosphorylation with T4 PNK, a preadenylated DNA adapter was ligated to the 3’ end. Samples were then run on NuPAGE gels and transferred to nitrocellulose membranes. After cutting out the region of nitrocellulose containing RNA–protein complexes, RNAs were removed from the membrane by Proteinase K digestion.

treatment_protocol: Transfection of siRNAs was performed using Cell Line 96-well Nucleofector Kit SF and 96-well Shuttle Device (Lonza).

tag: cell type

value: Ovarian Somatic Cells (OSC)

tag: variation

tag: chip/clip antibody

value: anti-Piwi antibody (Satio et al., Genes Dev. 2006)

tag: molecule subtype

value: immunoprecipitated RNA


title: mouse liver-232-NR,RNA-s102796-001-007_NR.fastq.R1


title: Piwi CLIP-seq rep1


title: mouse liver-289-HFD,RNA-s102796-001-005_HFD.fastq.R1


title: mouse liver-206-Chow,RNA-s102796-001-011_Chow.fastq.R1


title: mouse liver-202-Chow,RNA-s102796-001-001_Chow.fastq.R1


title: mouse liver-247-LC,RNA-s102796-001-013_LC.fastq.R1


title: mouse liver-234-NR,RNA-s102796-001-012_NR.fastq.R1


title: mouse liver-291-HFD,RNA-s102796-001-015_HFD.fastq.R1


title: Piwi-IP smRNA-seq

data_processing:
4nt random nucleotides and adapter sequences were removed from the obtained reads using Cutadapt, and reads out of 20-35nt size range were removed for further analysis. Obtained reads were mapped to the Drosophila genome (dm3) using Bowtie, allowing up to single mismatch. Genome-mapped reads were further mapped to TE consensus sequences from Repbase using Bowtie, allowing uniquely mapped reads. The reads were normalized to RPM by the number of Drosophila genome-mapped reads.
processed data files format and content: Normalized or raw read counts mapped to each transposon consensus sequence. Only reads mapped to sense direction was counted for CLIP analysis, and only reads mapped to antisense direction was counted for smRNA analysis (Tab delimited format, txt).
Genome_build: dm3

extract_protocol: Piwi was immunoprecipitated from confluent OSCs in 9-cm dishes, and Piwi-associated RNA was isolated from the immunopurified complex by phenol-chloroform extraction and ethanol precipitation.

value: small RNA


title: Nxf2 CLIP-seq SMInput from Piwi-KD sample rep2

source_name: Piwi&endoNxf2-KD OSC (myc-Nxf2 expressing stable line)

value: RNAi Piwi, Nxf2

value: size-selected RNA


title: mouse liver-259-Combi,RNA-s102796-001-004_Combi.fastq.R1

value: HFD+LC+NR = HFD+Combi


title: Piwi CLIP-seq SMInput


title: Nxf2 CLIP-seq from Control(siEGFP)-KD sample rep2

source_name: EGFP&endoNxf2-KD OSC (myc-Nxf2 expressing stable line)

value: RNAi EGFP, Nxf2

value: anti-myc antibody (9E10, DSHB)


title: Nxf2 CLIP-seq from Piwi-KD sample rep1


title: mouse liver-245-LC,RNA-s102796-001-003_LC.fastq.R1


title: mouse liver-229-NR,RNA-s102796-001-002_NR.fastq.R1


title: Nxf2 CLIP-seq SMInput from Piwi-KD sample rep1


title: Nxf2 CLIP-seq from Piwi-KD sample rep2


title: RNA-seq from Panx-KD rep2

data_processing:
Bowtie 2.2.9 was used with the default parameters to map sequences to the Drosophila genome (dm3). Reads mapped to the genome were then mapped to the Drosophila melanogaster TE consensus sequence from Repbase, using Bowtie 1.0.1, and those that were uniquely mapped were extracted. Expression levels (RPKM) of TEs were calculated using reads that were mapped to the TE consensus sequence. Expression levels (RPKM) of coding genes were calculated using TopHat 2.0.14 and DESeq2.
processed data files format and content: Normalized or raw read counts mapped to each transposon consensus sequence. Only reads mapped to sense direction was counted for CLIP analysis, and only reads mapped to antisense direction was counted for smRNA analysis (Tab delimited format, txt).
Genome_build: dm3

source_name: Panx-KD OSC

extract_protocol:
Total RNAs were isolated with the Isogen reagent (NipponGene). Poly(A)+ RNAs were obtained using Oligo-dT beads.
Poly(A)+ RNA libraries were prepared as described (Ohtani et al., 2013).

value: RNAi Panx

value: total RNA


title: Nxf2 CLIP-seq SMInput from Control(siEGFP)-KD sample rep2


title: RNA-seq from Control(EGFP)-KD rep2

source_name: EGFP-KD OSC

value: RNAi EGFP


title: Nxf2 CLIP-seq from crosslink pos sample rep1

source_name: OSC (myc-Nxf2 expressing stable line)


title: Nxf2 CLIP-seq SMInput from crosslink pos sample


title: RNA-seq from Nxf2-KD rep2

source_name: Nxf2-KD OSC

value: RNAi Nxf2


title: Nxf2 CLIP-seq from crosslink neg sample rep1


title: Nxf2 CLIP-seq from crosslink pos sample rep2


title: ChIP-seq input from Panx-KD

library_source: genomic

data_processing:
Adapters were removed from the reads using Cutadapt, and subsequent reads shorter than 50 nt were discarded. ChIP-seq reads were mapped to the Drosophila genome (dm3) using Bowtie 1.0. Reads mapped to the genome were extracted and mapped to Drosophila melanogaster TE consensus sequences from Repbase, permitting only those that mapped uniquely to the TE consensus sequence. For peak calling of H3K9me3 signals, MACS-1.4.2 was used with the default parameters.
processed data files format and content: Peaks called using MACS-1.4.2 (bed format)
Genome_build: dm3

molecule: genomic DNA

extract_protocol:
OSC cells were fixed, lysed, and their nuclei isolated using truChIP Chromatin Shearing Kits (Covaris) according to the manufacturer’s instructions. DNA was sonicated to ~300 bases using Bioruptor (Cosmobio), then diluted with ChIP buffer. IP was performed using 3 μg of antibodies on Dynabeads-Protein G beads. Samples were reverse cross-linked, treated with RNase and Proteinase K, and DNA was extracted.
DNA fragments from the ChIP experiment were sheared to ~200 bases using Covaris S220 (Covaris), and were used for library preparation with the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) following the manufacturer’s protocol.

value: genomic DNA


title: H3K9me3 ChIP-seq from Panx-KD rep1

value: anti-H3K9me3 antibody (61013, Active motif)


title: H3K9me3 ChIP-seq from Panx-KD rep2


title: H3K9me3 ChIP-seq from Nxf2-KD rep1


title: RNA-seq from Panx-KD rep1


title: Nxf2 CLIP-seq from Control(siEGFP)-KD sample rep1


title: Nxf2 CLIP-seq from crosslink neg sample rep2


title: RNA-seq from Piwi-KD rep1

source_name: Piwi-KD OSC

value: RNAi Piwi


title: Nxf2 CLIP-seq SMInput from crosslink neg sample


title: RNA-seq from Nxf2-KD rep1


title: 11T-1-RX-CrePlus-Plus-WT-1_Aug18: wt transformed cells variant#11 repl1 4-OHT

status: Public on Jun 17 2019

city: Buffalo

first: Katerina

last: Gurova

state: NY

address: Elm and Carlton Str

department: Cell Stress Biology

country: USA

institute: Roswell Park Cancer Institute

data_processing:
Basecalls performed using bcl2fastq v2.20.0.422
RNASeq reads are mapped to mm10 reference genome and refGene annotation database using TopHat 2.1.1
Gene expression quantification is done using featureCounts v1.4.6 with –fracOverlap 1
Genome_build: mm10
Supplementary_files_format_and_content: comma separated file with raw reads count

source_name: Tail Fibroblasts

growth_protocol: For the generation of primary fibroblasts from Ssrp1++lCreERT2+/+, Ssrp1fl/flCreERT2+/+, and Ssrp1fl/+CreERT2+/+ mice, approximately 2 cm piece of the tail tip was collected and digested using 2mg/ml Collagenase A. Isolated cells were maintained in F-12K medium containing 10% FBS and 1% AA. Primary fibroblasts were infected with genetic suppressor element 56 (GSE56) virus and subsequently selected with 500 μg/ml neomycin (G418 Sulfate) to generate immortalized cells. For the generation of transformed fibroblasts, the immortalized fibroblasts were infected with oncogenic H-RASV-12 virus and selected with bleomycin (30 ug/ml). Immortalized and transformed cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were kept in a sterile humidified tissue culture incubator at 37°C and 5% CO2 for 4 to 5 days to allow cells to adhere to the plate.

extract_protocol:
Monarch® Total RNA Miniprep Kit-T2010S
KAPA RNA HyperPrep with RiboErase
NovaSeq S1 100 PE

treatment_protocol: For the excision of Ssrp1 in vitro, primary, immortalized, or transformed cells (1x106) were plated in 150 mm plates. The cells were treated with 2 μM 4-hydroxytamoxifen (4-OHT) for 96 h (primary cells) or 120 h (immortalized and transformed cells). The medium was replaced every 48 h with fresh 2 μM 4-OHT containing medium. The excision of Ssrp1 was confirmed at both the DNA and protein level.

tag: strain

tag: phenotype

value: Transformed

tag: tissue

value: Tail Fibroblasts

tag: treatment

value: Tamoxifen


title: RNA-seq from Control(EGFP)-KD rep1

value: Ovarian Somatic Cells (OSC)


title: H3K9me3 ChIP-seq from Nxf2-KD rep2


title: 11T-2-RX-CrePlus-Plus-WT-2_Aug18: wt transformed cells variant#11 repl2 4-OHT


title: 11T-2-CrePlus-Plus-WT-2_Aug18: wt transformed cells variant#11 repl2 control

value: Untreated


title: 3I-R1_Aug18: flox immortalized cells variant#3 repl1 4-OHT

value: Immortalized


title: ChIP-seq input from Nxf2-KD


title: 11-1-CrePlus-Plus-WT-1_June18: wt primary cells variant#11 repl1 control

value: Primary


title: 11-2-RX-CrePlus-Plus-WT-2_June18: wt primary cells variant#11 repl2 4-OHT


title: 11I-1-CrePlus-Plus-WT-1_Aug18: wt immortalized cells variant#11 repl1 control


title: 3T-2_June18: flox transformed cells variant#3 repl2 control


title: 4-2-RX-CrePlus-Plus-fl-fl-1_June18: flox primary cells variant#4 repl2 4-OHT


title: 11-1-RX-CrePlus-Plus-WT-1_June18: wt primary cells variant#11 repl1 4-OHT


title: 4-1-CrePlus-Plus-fl-fl-1_June18: flox primary cells variant#4 repl1 control


title: 11I-1-RX-CrePlus-Plus-WT-1_Aug18: wt immortalized cells variant#11 repl1 4-OHT


title: Nxf2 CLIP-seq SMInput from Control(siEGFP)-KD sample rep1

value: size-selected RNA


title: 3T-R-1_June18: flox transformed cells variant#3 repl1 4-OHT


title: 4-1-RX-CrePlus-Plus-fl-fl-1_June18: flox primary cells variant#4 repl1 4-OHT


title: 3T-1_June18: flox transformed cells variant#3 repl1 control


title: 3T-R-2_June18: flox transformed cells variant#3 repl2 4-OHT


title: 11T-1-CrePlus-Plus-WT-1_Aug18: wt transformed cells variant#11 repl1 control


title: 11I-2-CrePlus-Plus-WT-2_Aug18: wt immortalized cells variant#11 repl2 control


title: 3I-1_June18: flox immortalized cells variant#3 repl1 control


title: 3I-R1_June18: flox immortalized cells variant#3 repl1 4-OHT


title: 3I-R2_Aug18: flox immortalized cells variant#3 repl2 4-OHT


title: 4-2-CrePlus-Plus-fl-fl-2_June18: flox primary cells variant#4 repl2 control


title: 11I-2-RX-CrePlus-Plus-WT-2_Aug18: wt immortalized cells variant#11 repl2 4-OHT


title: RNA-seq_flox primary cells variant#8  4-OHT

status: Public on Jun 14 2019

description:
CountsSubread.csv
name in processed data file:
8_Rx_June17

extract_protocol:
 Qiagen miRNAeasy kit
 Illumina TruSeq Stranded RNA with RiboZero Gold, NextSeq HighOutput 75 SR

tag: cell status

tag: genotype


title: 3I-2_June18: flox immortalized cells variant#3 repl2 control


title: RNA-seq_flox primary cells variant#13 control

description:
CountsSubread.csv
name in processed data file:
13_Ctrl__June17


title: RNA-seq_flox transformed cells variant#2  4-OHT

description:
CountsSubread.csv
name in processed data file:
2T_Rx_June17


title: RNA-seq_flox primary cells variant#8 control

description:
CountsSubread.csv
name in processed data file:
8_Ctrl__June17


title: RNA-seq_flox transformed cells variant#3  4-OHT

description:
CountsSubread.csv
name in processed data file:
3T_Rx_June17


title: 11-2-CrePlus-Plus-WT-2_June18: wt primary cells variant#11 repl2 control


title: RNA-seq_flox primary cells variant#13  4-OHT

description:
CountsSubread.csv
name in processed data file:
13_Rx_June17


title: 3I-R2_June18: flox immortalized cells variant#3 repl2 4-OHT


title: RNA-seq_flox immortalized cells variant#3  4-OHT

description:
CountsSubread.csv
name in processed data file:
3I_Rx_June17


title: RNA-seq_flox transformed cells variant#3  control

description:
CountsSubread.csv
name in processed data file:
3T_Ctrl_June17


title: RNA-seq_flox immortalized cells variant#2  4-OHT

description:
CountsSubread.csv
name in processed data file:
2I_Rx_June17


title: RNA-seq_flox transformed cells variant#2 control

description:
CountsSubread.csv
name in processed data file:
2T_Ctrl_June17


title: RNA-seq_flox immortalized cells variant#3  control

description:
CountsSubread.csv
name in processed data file:
3I_Ctrl_June17


title: SSRP1_ChIP-seq 3T-SSRP1_7618

status: Public on Jun 12 2019

data_processing:
Basecalls performed using CASAVA version 1.8.2
ChIP-seq reads were aligned to the mm10 genome assembly using bwa 0.7.15
PCR duplicate reads were marked using picard 1.97
Genome_build: mm10
Supplementary_files_format_and_content: big wig files were generated using deeptools 2 with RPKM normalization, score represents normalized coverage

source_name: transformed mouse skin fibroblasts, variant #3

growth_protocol: Cells were maintaned in 10% FBS, 1% pen/strep DMEM

extract_protocol: SimpleChip Kit #9003 (Cell Signaling) was used for preparation DNA samples for library

treatment_protocol: 4X150 mm plates of each cell line were used for preparation of DNA samples for ChIP-Seq

tag: cell culture viarians

tag: chip-antibody

value: SSRP1 (BioLegend, 609702, 10D1)


title: H3_ChIP-seq 3T-H3_7618

value: transformed mouse skin fibroblasts, variant #3

value: Histone H3 (Cell Signaling, 9003,D2B12)


title: input DNA (MNase-seq) 2T-nDNA-input

description: input DNA (MNase-seq)

source_name: transformed mouse skin fibroblasts, variant #2


title: SSRP1_ChIP-seq 2T-SSRP1_6818

value: transformed mouse skin fibroblasts, variant #2


title: SSRP1_ChIP-seq 2I-SSRP1_7618

source_name: immortalized mouse skin fibroblasts, variant # 2


title: input DNA (MNase-seq) 2T-input_5918

description: input DNA (MNase-seq)


title: SSRP1_ChIP-seq 2T-SSRP1_5918


title: H3_ChIP-seq 3I-H3

source_name: immortalized mouse skin fibroblasts, variant # 3


title: SSRP1_ChIP-seq 2I-SSRP1

value: immortalized mouse skin fibroblasts


title: H3_ChIP-seq 3T-H3


title: SSRP1_ChIP-seq 3I-SSRP1

value: immortalized mouse skin fibroblasts, variant # 3


title: H3_ChIP-seq 2T-H3_6818


title: H3_ChIP-seq 2T-H3


title: input DNA (MNase-seq) 3T-nDNA-input

description: input DNA (MNase-seq)


title: atfs1b

status: Public on Nov 21 2018

city: Montreal

first: Jeremy

middle: Michael

last: Van Raamsdonk

state: QC

address: 1001 Decarie Boulevard

department: Neurology and Neurosurgery

institute: McGill University

data_processing:
Libraries were sequenced using 1x75 bp sequencing on the Illumina NextSeq 500 platform at the Van Andel Research Institute.  Data were demultplexed using bcl2fastq v 2.18.
Read quality was assessed using FASTQC v. 0.11.5 and aligned first to the E. coli K-12 genome to assess contamination, then one-pass aligned to WBcel235 C. elegans genome using STAR v. 2.5.2b with default parameters and --outReadsUnmapped None. The STAR genome index was generated with the corresponding Ensembl WBcel235 build 89 GTF annotations integrated.
Transcript abundances were quantified using the --quantMode GeneCounts option enabled during alignment. Differential gene expression analysis was performed using the quasi-likelihood framework in edgeR package v. 3.20.1 in R v. 3.4.1. The model used for DGE accounts for sequencing batch and genotype and is as follows: design <- model.matrix(~seqbatch + genotype). Gene names, gene IDs, and predicted function annotations were downloaded using the biomaRt package v. 2.34.0.
Filtered CPM were filtered keeping genes that had CPM > 2 in at least 3 samples. Raw CPM are unfiltered.
Genome_build: WBcel235
Supplementary_files_format_and_content: Text file containing filtered gene counts per million matrix (CPM) from all samples.

organism: Caenorhabditis elegans

source_name: Whole worm

growth_protocol: NGM plates, 20 degrees Celsius, OP50 bacteria

extract_protocol:
See Schaar et al. 2015 PLoS Genetics for RNA isolation protocol
Sequencing libraries were prepared using the Kapa Biosystems stranded mRNA-Seq kit for the Illumina platform.

value: Whole worm

tag: developmental stage

value: prefertile young adult

tag: sequencing batch


title: atfs1r


title: H3_ChIP-seq 2I-H3

value: immortalized mouse skin fibroblasts, variant # 2


title: IgG-ChIP-seq 2T-IgG_6818

description: IgG-ChIP-seq


title: H3_ChIP-seq 2T-H3_5918


title: NA238r


title: SSRP1_ChIP-seq 3T-SSRP1


title: input DNA (MNase-seq) 2I-nDNA-input

description: input DNA (MNase-seq)

source_name: immortalized mouse skin fibroblasts, variant #3


title: nuo6_6


title: NA238b


title: atfs1bl


title: RNA-seq_flox immortalized cells variant#2 control

description:
CountsSubread.csv
name in processed data file:
2I_Ctrl_June17


title: SSRP1_ChIP-seq 2I-SSRP1_6818


title: NA238bl


title: H3_ChIP-seq 2I-H3_6818


title: SSRP1_ChIP-seq 2T-SSRP1


title: nuo6_3


title: nuo6hif12


title: nuo6_5


title: nuo6_4


title: nuo6hif13


title: atfs1et174


title: atfs1et175


title: atfs1et154


title: atfs1et172


title: atfs1et171


title: hif12


title: atfs1et155


title: nuo6_2


title: atfs1et173


title: WTr


title: WTbl


title: WTb


title: atfs1et156


title: atfs1et153


title: hif13


title: atfs1DEL2


title: hif11


title: atfs1et176


title: atfs1DEL1


title: WT_7


title: atfs1DEL3


title: nuo6_1


title: atfs1et152


title: WT_8


title: WT_9


title: WT_5


title: WT3


title: WT_6


title: WT1


title: WT_4


title: WT2


title: IgG CBL0137

status: Public on Jul 20 2018

data_processing:
Basecalls performed using CASAVA version 1.9
ChIP-seq reads were aligned to the hg19 genome assembly using bwa sampe with default parameters
BigWig files and heatmap/profiles are generated using deeptools
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files from deeptools

source_name: HT1080 cells

growth_protocol: DMEM 5% FBS

extract_protocol:
Samples for ChIP Sequencing were prepared using SimpleChIP Kit # 9003 from Cell Signaling Technology without modifications. Cells HT1080, tail fibroblasts from SSRP1 KO mice were grown to 80% confluence, fixed with 1% formaldehyde for 10 min and  protocol for this kit was followed: chromatin was digested with Micrococcal Nuclease into 150-900 bp DNA/protein fragments, antibodies specific to H3 ( provided by kit) and to H3 acetyl K27 ( ab4729, Abcam) were added and the complex co-precipitated and was captured by protein G magnetic beads. Cross-links were reversed, and DNA was purified for sequencing.
20ng of Chromatin-immunoprecipitated (ChIP) DNA is used to generate a library for Next generation sequencing using the ThruPLEX DNA seq kit (Rubicon Genomics, Inc.) as per manufacturer’s instructions.  ThruPLEX uses stem-loop adapters to construct high quality libraries in a fast and efficient workflow. In the first step, Template Preparation, the DNA is repaired and yields molecules with blunt ends. Next, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end.  In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. The library is quantitated using quantitative PCR (KAPA Biosystems), normalized, and pooled in an equimolar fashion.  Pooled libraries are loaded to an individual lane of a HiSeq High Output v3 flow cell, using an Illumina cBot, for 2 x 75 PE and 75bp single end  sequencing on a NextSeq sequencer according to the manufacturer's recommended protocol (Illumina Inc.).

treatment_protocol: 6 hours, 3uM of CBL0137

tag: cell line

value: HT1080 cells

value: 3 uM CBL0137, 6 hours

tag: antibody

value: IgG


title: H3K27ac control replicate 1

value: no

value: H3K27ac antibody


title: H3K27ac CBL0137


title: H3 CBL0137

value: H3 antibody


title: H3 control replicate 2


title: H3K27ac control replicate 2


title: H3 control replicate 1


title: dsNbr_Flag-Ago3-smRNA-seq

status: Public on Mar 01 2018

description: RNA (immunoprecipitated)

data_processing:
Adapter sequences were removed from the obtained reads, and the reads were mapped to the silkworm reference genome using Bowtie, allowing no mismatches. Genome mapped reads were extracted and aligned to B. mori transposon consensus sequences (Kind gift from Dr. S. Kawaoka) using Bowtie, allowing up to two mismatches.
Genome_build: silkworm reference genome (downloaded from the Silkworm Genome Research Program Database; http://sgp.dna.affrc.go.jp/data/integretedseq.txt.gz).
Supplementary_files_format_and_content: space-delimited text files include count of reads mapped to each transposable element (TE) consensus sequence in sense and antisense direction.

organism: Bombyx mori

source_name: BmN4 cells

growth_protocol: BmN4 cells were grown at 27 degrees on Ex-Cell medium

extract_protocol:
RIP-seq: RNAs were eluted from the PAPI immunoprecipitates by phenol-chloroform after protease K treatment and precipitated with ethanol. RNAs were resolved by denaturing PAGE, and 65- to 100-nt-long RNAs were eluted from gels.
smRNA-seq: Immunoprecipitation of Flag-Siwi and Flag-Ago3 was carried out as described previously (Nishida et al., 2015). RNAs were eluted from the immunoprecipitates by phenol-chloroform after protease K treatment, and precipitated with ethanol. RNAs of 23–35 nts in length were eluted from the gels and used to generate small RNA libraries.
The libraries were generated with NEBNext Small RNA Library Sample Prep Set (New England BioLabs (NEB), E7330), as described previously (Nishida et al., 2015).

treatment_protocol:
RNAi-KD: BmN4 cells were transfected with 2 µg of dsRNAs (per 3 × 10^5 cells) using FuGENE HD (Promega).
Flag-Siwi/Ago3 expression: To exogenously express proteins in BmN4 cells, the cells were transfected with 2 µg of plasmids (per 6 × 10^5 cells) using FuGENE HD.

value: BmN4 cells

value: anti-DDDDK-tag (Flag) (MBL:M185-3)


title: dsTrim_Flag-Siwi-smRNA-seq

description: RNA (immunoprecipitated)


title: dsLuc_Flag-Siwi-smRNA-seq

description: RNA (immunoprecipitated)


title: dsZuc_PAPI-RIP-seq

description: RNA (immunoprecipitated)

value: anti-PAPI (generated in this study)


title: dsTrim_Flag-Ago3-smRNA-seq

description: RNA (immunoprecipitated)


title: dsLuc_Flag-Ago3-smRNA-seq

description: RNA (immunoprecipitated)


title: dsNbr_Flag-Siwi-smRNA-seq

description: RNA (immunoprecipitated)


title: Aclacinomycin A (AclA) replicate 2

status: Public on Mar 13 2018

data_processing:
Basecalls performed using CASAVA version 1.9
ChIP-seq reads were aligned to the hg19 genome assembly using bwa sampe with default parameters
Broad peaks were called using MACS2 with default parameters
BigWig files and heatmap/profiles are generated using deeptools
Genome_build: hg19
Supplementary_files_format_and_content: Bed files with peak calling results from MACS2, bigWig files from deeptools using RPKM normalization.

source_name: HT1080_Aclacinomycin A (AclA)

extract_protocol:
ChIP-with SSRP1 antibody
20ng of Chromatin-immunoprecipitated (ChIP) DNA is used to generate a library for Next generation sequencing using the ThruPLEX DNA seq kit (Rubicon Genomics, Inc.) as per manufacturer’s instructions.  ThruPLEX uses stem-loop adapters to construct high quality libraries in a fast and efficient workflow. In the first step, Template Preparation, the DNA is repaired and yields molecules with blunt ends. Next, stem-loop adaptors with blocked 5’ ends are ligated with high efficiency to the 5’ end of the genomic DNA, leaving a nick at the 3’ end.  In the final step, the 3’ ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. The library is quantitated using quantitative PCR (KAPA Biosystems), normalized, and pooled in an equimolar fashion.  Pooled libraries are loaded to an individual lane of a HiSeq High Output v3 flow cell, using an Illumina cBot, for 2 x 100 PE sequencing on a HiSeq2500 sequencer according to the manufacturer's recommended protocol (Illumina Inc.).

treatment_protocol: 1.5 hours, 3uM of CBL0137, 0.3uM of CBL0100 or 3uM of aclacinomycin A

tag: treated with

value: Aclacinomycin A

tag: concentration

tag: time of treatment

tag: chip antibody

value: SSRP1 antibody

tag: chip antibody vendor

value: BioLegend

tag: chip antibody clone

tag: chip antibody cat#


title: HeLaS3 control  1st replicate

status: Public on Jul 25 2018

type: RNA

description:
replicate 1
SAMPLE 9

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: Standard Illumina scanning protocol

data_processing: The data were summarized and quantile normalized by using  GenomeStudio 1.9.0

label: biotin

source_name: HeLaS3 control

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

growth_protocol: standard, DMEM + 5% FBS + antibiotics

extract_protocol: TRIZOL reagent

treatment_protocol: 6 hours of CBL0137, 0, 0.3, 1 and 3 microM for MM1.S; 0 and 3microM for HeLa, controls - plain medium

value: HeLa S3


title: HeLaS3 control  2nd replicate

description:
Replicate 2
SAMPLE 10


title: control replicate 1

value: none (control)


title: CBL0100 replicate 1


title: CBL0137 replicate 2