title: PRO-Seq_RPB12_Untreated_rep2

description:
PRO-Seq_RPB12_Untreated_plus.bw
PRO-Seq_RPB12_Untreated_minus.bw

data_processing:
Library strategy: PRO-Seq
PRO-seq raw data were processed using a custom pipeline that builds on published workflows with minor modifications. Briefly, ribosomal reads were first removed by mapping to one copy of the mouse rDNA sequence. After adapter trimming and quality control with cutadapt and RseQC separately, clean reads were then aligned to a concatenated genome comprised of mouse mm10 and fly dm6 assemblies using bowtie2 with options (-very-sensitive-local) as described in the ChIP-seq section.
Only reads that were properly paired and uniquely aligned were retained for subsequent processing. Reads that mapped to the mouse genome were normalized to total reads originating from Drosophila spike-in cells, and the normalization factor was calculated as previously described (Jiang et al., 2020).
Plus- and minus-strand were assigned to uniquely mapped reads using SAMtools, and the final BAM files were converted to bigwig, separated by strand, and normalized to spike-in controls using bamCoverage from deeptools. Finally, bigWig score tracks for visualization prepared for different biological replicates but for the same treatment were merged to an averaged signal using the mean operator from the WiggleTools (v1.2) package (Zerbino, Johnson, et al. 2014).
Then raw read counts were calculated per gene, in a strand-specific manner, based on annotations described in the Gene list definition section below, using featureCounts (Liao et al., 2014).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files

growth_protocol: For Pro-seq, wild-type and Pol II subunit degron cells were cultured in DMEM-KO (Thermo Fisher, Cat#10829018) supplemented with 15% FBS (Gibco, Cat#10099141C), 1× penicillin/ streptomycin (Gibco, Cat#15140122), 1 × L-glutamine (Gibco, Cat#25030081), 1 × non-essential amino acids (Gibco, Cat#11140050), 1 × nucleoside (Millipore, Cat#ES-008-D), 3 μM CHIR99021 (Selleck, Cat#S1263), 0.5 μM PD0325901 (Selleck, Cat#S1036), 143 uM 2-mercaptoethanol (Thermo Fisher, Cat#M3148) and maintained at 37℃ cell incubator with 5% CO2. The Schneider 2(S2) cells were cultured in complete Schneider’s Drosophila Medium (Invitrogen, Cat#21720024) supplemented with 10% FBS (CellMax, Cat#SA311.02) at room temperature.

extract_protocol:
1-Biotin run-on was applied according to the protocol of Dig et al. (Dig et al., 2016). Total RNA was extracted using TRIzol reagent and fragmented by base hydrolysis. The nascent RNA labeled by Biotin were enriched with streptavidin beads and extracted using TRIzol reagent again.
The enriched RNA was reverse transcribed into first-strand DNA using SuperScript IV reverse transcriptase kit (Invitrogen, 18090010) with N9 random primer. And RNA was eliminated by adding 2μl 1M NaOH and heating at 98℃ for 20min. DNA was purified with Phenol/Chloroform and precipitated with Ethanol. Finally, the DNA samples were subjected to TELP library construction (Peng et al., 2015). 200bp-400bp DNA libraries were gel-selected, purified, and subjected to HiSeq X-ten PE150 sequencing.
TELP libraries

treatment_protocol: Cells were treated with 2 μg/ml Doxycycline for 12 h to induce OsTir1 expression, then treated with or without 500 uM IAA for 1 h to induce target protein degradation. Cell nuclei were isolated according to the protocol of Dig et al. (Dig et al., 2016), and 3% S2 cells were added during the cell permeabilization.

value: RPB12 untreated mESCs


title: PRO-Seq_RPB12_IAA_rep2

status: Public on Aug 09 2022

type: SRA

city: Beijing

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

department: School of Life Sciences

country: China

institute: Peking University

description:
PRO-Seq_RPB12_IAA_plus.bw
PRO-Seq_RPB12_IAA_minus.bw

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

source_name: mouse embryonic stem cells (V6.5)

tag: cell line

value: mouse embryonic stem cells (V6.5)

tag: treatment

value: RPB12 degron 1h mESCs


title: SGE+BEV_2

type: RNA

city: Okayama

first: Yasuhiko

last: Hattori

address: 2-5-1 Shikata-cho, Kita-ku

country: Japan

institute: Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

hyb_protocol: Labeled probes were hybridized to the Agilent SurePrint G3 Mouse Gene Expression 8x60K and scanned.

scan_protocol: Expression data were extracted from image files produced on an Agilent microarray scanner.

data_processing: The scanned images were analyzed with Agilent Feature Extraction 12.1.1.1. Genechip analysis was performed using GeneSpring GX 14.9.1 software (Agilent).

label: biotin

source_name: mouse brain, Ad-SGE-REIC, bevacizumab 2

label_protocol: Amplification and biotin labeling of fragmented cDNA was performed using NuGEN Ovation Pico WTA System V2 (NuGEN Technologies Inc., San Carlos, CA).

extract_protocol: Approximately 40 mg of brain tumor samples were excised cleanly from each mouse, and RNA was extracted using TRIzol (Life Technologies) and an RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Samples were analyzed using the SurePrint G3 Mouse GE Microarray 8x60K Ver2.0 (Agilent, Tokyo, Japan).

treatment_protocol: Orthotopic U87ΔEGFR xenograft mouse models treated with the control, Ad-SGE-REIC, or combination of bevacizumab and Ad-SGE-REIC were euthanized 18 days after tumor implantation (n = 3/treatment).

tag: tissue

value: brain

tag: gender

value: female

value: with Ad-SGE-REIC and bevacizumab


title: ChIP-seq-03-SPT6-SPT6AID-Aux-2h

city: Chicago

first: Yuki

last: Aoi

state: IL

address: 303 E Superior St. SQBRC 7-300

department: Biochemistry & Molecular Genetics

country: USA

institute: Northwestern University

description: ChIP-seq SPT6

library_source: genomic

data_processing:
For ChIP-seq, single-end raw reads trimmed by cutadapt 1.14 (Martin, 2011) were aligned to a concatenated genome comprised of human hg38 and mouse mm10 assemblies, using bowtie 2.2.6 (Langmead and Salzberg, 2012) with --sensitive option. The aligned reads with MAPQ ≥ 30 were extended to 150 bp and read counts were normalized to total reads aligned to spike-in genome. The bigwig files with normalized counts were generated using deepTools 3.1.1 bamCoverage (Ramírez et al., 2016).
Assembly: hg38
Supplementary files format and content: bigWig files contain spike-in normalized ChIP-seq signal.
Supplementary files format and content: bed files contain coordinates of genes or pause sites that were used in this study.

molecule: genomic DNA

organism: Homo sapiens

growth_protocol: Human DLD-1 cells that constitutively express OsTIR1 were cultured at 37°C in DMEM (Sigma #D6429) supplemented with 10% FBS (Sigma #F2442), 1% Pen-Strep (Gibco #15140-122) in CO2 incubators.

extract_protocol:
For ChIP-seq in human DLD1 cells, ~30 million cells were crosslinked with 1% paraformaldehyde in PBS for 10 min at r.t. Spike-in mouse embryonic fibroblast fixed cells were added to samples. Chromatin was sonicated with the Covaris E220. Immunoprecipitations were carried out with antibodies and Dynabeads Protein G.
For ChIP-seq, DNA libraries were prepared by the HTP Library Preparation Kit for Illimina (KAPA).

treatment_protocol: To induce degradation of AID-tagged proteins, 500 µM auxin was added to culture.

tag: cell type

value: Colorectal adenocarcinoma

tag: genotype

value: SPT6-AID OsTIR1

value: Auxin 2h

tag: chip antibody

value: SPT6 (Cell signaling # 15616)


title: control_3

source_name: mouse brain, control 3

value: with PBS


title: SGE_3

source_name: mouse brain, Ad-SGE-REIC 3

value: with Ad-SGE-REIC


title: SGE_2

source_name: mouse brain, Ad-SGE-REIC 2


title: SGE+BEV_1

source_name: mouse brain, Ad-SGE-REIC, bevacizumab 1


title: SGE_1

source_name: mouse brain, Ad-SGE-REIC 1


title: control_1

source_name: mouse brain, control 1


title: SGE+BEV_3

source_name: mouse brain, Ad-SGE-REIC, bevacizumab 3


title: control_2

source_name: mouse brain, control 2


title: ChIP-seq-03-SPT6-SPT6AID-Veh-2h

description: ChIP-seq SPT6

value: Vehcle 2h


title: ChAR-Seq_RPB1_IAA_0h_rep1

description:
ChAR-Seq_RPB1_IAA_0h_plus.bw
ChAR-Seq_RPB1_IAA_0h_minus.bw

data_processing:
Library strategy: ChAR-Seq
ChAR-seq raw data were processed using a custom pipeline that builds on published workflows with minor modifications. Briefly, ribosomal reads were first removed by mapping to one copy of the mouse rDNA sequence. After adapter trimming and quality control with cutadapt and RseQC separately, clean reads were then aligned to a concatenated genome comprised of mouse mm10 and fly dm6 assemblies using bowtie2 with options (-very-sensitive-local) as described in the ChIP-seq section.
Only reads that were properly paired and uniquely aligned were retained for subsequent processing. Reads that mapped to the mouse (or human) genome were normalized to total reads originating from Drosophila spike-in cells, and the normalization factor was calculated as previously described (Jiang et al., 2020).
Plus- and minus-strand were assigned to uniquely mapped reads using SAMtools, and the final BAM files were converted to bigwig, separated by strand, and normalized to spike-in controls using bamCoverage from deeptools. Finally, bigWig score tracks for visualization prepared for different biological replicates but for the same treatment were merged to an averaged signal using the mean operator from the WiggleTools (v1.2) package (Zerbino, Johnson, et al. 2014).
Then raw read counts were calculated per gene, in a strand-specific manner, based on annotations described in the Gene list definition section below, using featureCounts (Liao et al., 2014).
Genome_build: mm10 or hg19
Supplementary_files_format_and_content: bigwig files

growth_protocol: For ChAR-seq, RPB1, RPB3, RPB6, RPB9 and piRPB3 degron cells were cultured in DMEM-KO (Thermo Fisher, Cat#10829018) supplemented with 15% FBS (Gibco, Cat#10099141C), 1× penicillin/ streptomycin (Gibco, Cat#15140122), 1 × L-glutamine (Gibco, Cat#25030081), 1 × non-essential amino acids (Gibco, Cat#11140050), 1 × nucleoside (Millipore, Cat#ES-008-D), 3 μM CHIR99021 (Selleck, Cat#S1263), 0.5 μM PD0325901 (Selleck, Cat#S1036), 143 uM 2-mercaptoethanol (Thermo Fisher, Cat#M3148) and maintained at 37℃ cell incubator with 5% CO2. The Schneider 2(S2) cells were cultured in complete Schneider’s Drosophila Medium (Invitrogen, Cat#21720024) supplemented with 10% FBS (CellMax, Cat#SA311.02) at room temperature.

extract_protocol:
Cells were collected and added to 10% S2 cells. The chromatin-associated RNA were extracted as reported (Dev M. Bhatt et al., 2012).
Chromatin-associated RNA were delivered to Novogene Company. The concentration, purity, and integrity of RNA were examined and followed by ribosomal RNA depletion and strand-specific library construction.
TELP libraries

treatment_protocol: Cells were treated with 2 μg/ml Doxycycline for 12 h to induce OsTir1 expression, then treated with or without 500 uM IAA for 1 h or 3h to induce target protein degradation.

value: RPB1 degron 0h mESCs

tag: fraction

value: Chromatin-associated RNA


title: ChIP-seq-02-RPB1N-SPT6AID-Aux-6h

description: ChIP-seq Pol II (total)

value: Auxin 6h

value: RPB1 NTD (Cell signaling # 14958S)


title: PRO-Seq_RPB11_IAA_rep2

description:
PRO-Seq_RPB11_IAA_plus.bw
PRO-Seq_RPB11_IAA_minus.bw

value: RPB11 degron 1h mESCs


title: PRO-Seq_RPB12_Untreated_rep1

value: mouse embryonic stem cells (V6.5)

value: RPB12 untreated mESCs


title: PRO-seq-04-PAF1AID-Aux2h-HS1h

description: PRO-seq

data_processing:
For PRO-seq, low-quality bases and adapters from 3’ ends of reads were removed using cutadapt 1.14 (Martin, 2011) requiring a read length of 16–36 bp. Reads derived from ribosomal RNA were filtered out by mapping reads on human and fly ribosomal DNA. The remaining reads were aligned using bowtie 2.2.6 with --very-sensitive option (Langmead and Salzberg, 2012), to a concatenated genome comprised of human hg38 and fly dm6 assemblies. The 5’ ends of aligned reads with MAPQ ≥ 30 were taken by bedtools genomecov 2.25 (Quinlan and Hall, 2010) with -strand and -5 options, and strands of the 5’ ends of reads were then swapped. Read counts were normalized to total reads aligned to the spike-in genome, then the strand-specific bigwig files were generated.
Assembly: hg38
Supplementary files format and content: bigWig files contain strand-specific, spike-in normalized PRO-seq signal.
Library strategy: PRO-seq

molecule: nuclear RNA

extract_protocol:
PRO-seq sample preparation was performed according to the previously described qPRO-seq protocol (Judd et al., 2020). ~1 million DLD-1 nuclei  mixed with spike-in Drosophila S2 nuclei were used for nuclear run-on assay.
For PRO-seq, adapter sequences previously described (Mahat et al., 2016) were used. PRO-seq libraries were prepared according to the previously described qPRO-seq protocol (Judd et al., 2020).

value: PAF1-AID OsTIR1

value: Auxin 2h HS 1h


title: ChAR-Seq_RPB1_IAA_0h_rep2


title: scRNAseq

city: Qingdao

first: Hai

middle: Xia

last: Qu

state: China

address: No. 5, Donghai Zhong Road

institute: Qingdao Municipal Hospital

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: GRCh38-1.2.0
Supplementary files format and content: Tab-separated values files and matrix files

source_name: hepatocellular carcinoma

extract_protocol: For scRNA-seq, freshly excised tissue was rinsed with RPMI 1640 medium (Thermo Fisher Scientific, Inc.) and cut into 1-2-mm pieces. The samples were incubated at 37°C for 40 min with digestive solution, containing 0.25% trypsin (Thermo Fisher Scientific, Inc.) and 10 μg/ml DNase I (Sigma-Aldrich; Merck KGaA) dissolved in 5% FBS (Thermo Fisher Scientific, Inc.). Samples were manually oscillated every 5 min, and filtered twice using a 40-μm nylon mesh (Thermo Fisher Scientific, Inc.). Erythrocytes were removed using 1X Red Blood Cell Lysis Solution (Thermo Fisher Scientific, Inc.).

value: hepatocellular carcinoma

tag: Sex

value: male

tag: age

value: 65 years old


title: PRO-Seq_RPB12_IAA_rep1

value: RPB12 degron 1h mESCs


title: PRO-Seq_RPB11_IAA_rep1


title: PRO-seq-04-PAF1AID-Veh2h-NHS1h

description: PRO-seq

value: Colorectal adenocarcinoma

value: Vehcle 2h NHS 1h


title: PRO-seq-04-PAF1AID-Veh2h-HS1h

description: PRO-seq

value: Vehcle 2h HS 1h


title: ChIP-seq-02-RPB1N-SPT6AID-Aux-0h

description: ChIP-seq Pol II (total)

value: SPT6-AID OsTIR1

value: Auxin 0h


title: ChIP-seq-02-RPB1N-SPT6AID-Aux-2h

description: ChIP-seq Pol II (total)

value: Auxin 2h


title: PRO-seq-04-PAF1AID-Aux2h-NHS1h

description: PRO-seq

value: Auxin 2h NHS 1h


title: PRO-Seq_RPB11_Untreated_rep2

description:
PRO-Seq_RPB11_Untreated_plus.bw
PRO-Seq_RPB11_Untreated_minus.bw

value: RPB11 untreated mESCs


title: small RNAs from rhino[-/-]

city: Vienna

first: Lisa

last: Baumgartner

address: Dr. Bohr-Gasse 3

country: Austria

institute: IMBA

data_processing:
reads were trimmed by removal of the adaptor sequences and to a length of 6-50 bases
reads were mapped to the Drosophila melanogaster TE consensus using Bowtie (release 1.3.0) with 3 mismatches for genome wide analysis
reads mapping to annotated rRNA, tRNA, snRNA, snoRNA loci and the mitochondrial genome were excluded from analyses
reads were normalized to 1Mio miRNA reads for piRNA analysis
Assembly: dm6
Supplementary files format and content: all mapping and unique mapping bigwig files for sense and antisense

organism: Drosophila melanogaster

source_name: ovary

growth_protocol: 25˚C, 12h dark/light

extract_protocol:
Argonaute-bound sRNAs were isolated according to Grentzinger et al. (2020)
Libraries were prepared by ligation of 3' and 5' barcoded adaptors containing 4 random nucleotides at the ends to reduce ligation biases followed by reverse transcription and amplification by PCR.

value: ovary


title: PRO-Seq_RPB11_Untreated_rep1

value: mouse embryonic stem cells (V6.5)


title: PRO-seq-02-SPT6AID-Veh2h-HS1h

description: PRO-seq


title: ChIP-seq-01-RPB1N-SPT6AID-Aux-2h

description: ChIP-seq Pol II (total)

value: RPB1 NTD (Cell signaling # 14958S)


title: small RNAs from CG2678[fs1] ovaries


title: PRO-seq-02-SPT6AID-Aux2h-HS1h

description: PRO-seq


title: PRO-seq-02-SPT6AID-Aux2h-NHS1h

description: PRO-seq


title: Ribo_6h_KCl_rep3

city: Boston

first: Erin

middle: Elizabeth

last: Duffy

state: MA

address: 200 Longwood Ave, Goldenson 405

department: Neurobiology

institute: Harvard Medical School

description: Ribosome-associated RNA

data_processing:
Library strategy: Ribo-seq
In an effort to capture the most complete picture of translation, including the potential translation of brain-specific lncRNAs, RNA-seq and Ribo-seq reads were mapped to the lncRNA knowledge base (lncRNAKB) annotation.68 This annotation includes experimental evidence of lncRNA expression across 31 solid human normal tissues, including the brain, providing a comprehensive resource of transcripts and transcript isoforms in the human brain. Sequencing reads were aligned using Hisat2 (version 2.1.0) to the H. sapiens genome (GRCh30) and transcriptome (lncRNAKB). Alignments and analysis were performed on the Orchestra2 high performance computing cluster through Harvard Medical School. Aligned bam files were sorted using Picard Tools (version 2.8.0), stranded bedGraphs were generated using STAR, and reads were quantified over annotated exons using HTSeq-count (version 0.9.1).
Sequencing adapters were removed using Cutadapt (version 1.14), trimmed fastq files were aligned to hg38 ribosomal RNA sequences, and unaligned reads were mapped to the hg38 genome and lncRNAKB transcriptome68 using STAR (version 2.7.3a) with standard settings and the following modified parameters: --clip5pNbases 3, --seedSearchStartLmax 15, --outSJfilterOverhangMin 30 8 8 8, --outFilterScoreMin 0, -outFilterScoreMinOverLread 0.66, -outFilterMatchNmin 0, -outFilterMatchNminOverLread 0.66, --outSAMtype BAM Unsorted. Aligned bam files were sorted using Picard Tools (version 2.8.0) and stranded bedGraphs were generated using STAR. The RibORF pipeline was run on each sample individually using standard parameters. Due to template switching during library preparation, reads contained three untemplated bases at the 3’ end that were not included in the alignment but added to the length of each read. Therefore, reads 30-33 nt in length (corresponding to RNA fragments 27-30 nt) were analyzed for three-nucleotide periodicity within known protein-coding ORFs (RefSeq). For each sample we selected only the read lengths for which at least 50% of the reads matched the primary ORF of known protein-coding genes in a meta-gene analysis. Samples with fewer than two read lengths passing filtering were removed from further analysis. Read lengths were offset-corrected and RibORF was used to predict ORFs with a minimum length of 8 AA and translation probability >0.7. After running the RibORF pipeline on each brain sample individually, information from RibORF output files was used to generate GTF and BED files for all ORFs identified in each sample. ORFs with lengths of zero and ORFs annotated as non-coding despite being detected in protein coding genes were eliminated. Using Bedtools version 2.27.1 and the GRCh38 primary assembly human genome file, DNA sequences were associated with each exon of each remaining ORF. ORFs that did not end in stop codon sequences (“TGA”,”TAA”,”TAG”) were eliminated. Using the R library micropan version 2.1, DNA sequences for each complete ORF were translated into protein sequences. Of note, ORFs with start codons “GTG” or “TTG” are translated with a Methionine as the initial amino acid despite these sequences not typically encoding methionine in other positions in a protein-coding DNA sequence, per existing literature on non-canonical start codon usage in translation. Finally, all remaining ORF information was collapsed into one table, and duplicate ORFs, defined as ORFs in the same genomic position with identical protein sequences, were eliminated. When eliminating duplicates, ORFs identified of the most common ORF type identified by RibORF were conserved, according to the following order of priority, from highest to lowest: canonical, truncation, extension, overlap, uORF, internal, external, polycistronic, readthrough, and non-coding ORFs. ORFs annotated as type “seqerror” were eliminated. After combining ORF outputs from all samples, ORFs that were only detected in one sample were eliminated. After the removal of singleton ORFs, duplicate ORFs were once again eliminated according to the same priority scheme, leaving only one entry for each ORF that was detected in at least two samples in the dataset. The LncRNAKB annotation was used to assign a specific ORF type to each ORF. In order to be designated a sORF, an ORF had to be 100 amino acids or less in length, and not fully overlap in-frame with a canonical protein-coding ORF.
Genome_build: hg38

source_name: Cultured Neurons

growth_protocol: The use of hESCs was approved by the Harvard Medical School Embryonic Stem Cell Research Oversight (ESCRO) Committee. We used the H9 NGN2 hESC line, harboring a doxycycline-inducible NGN2 construct in the AAVS1 locus, referred to here as H9 NGN267.  We collected human neurons from three independent differentiation cohorts, and each replicate exhibited characteristic gene expression patterns reported previously.

extract_protocol:
Ribosome profiling was performed using a protocol modified from McGlincy et al. Frozen brain tissue (~80 mg) was thawed on ice. Each sample was dounced 15x in 400 µL ice cold lysis buffer: 20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide (Sigma). The lysate was further sheared using a 26-gauge syringe. The lysate was clarified by centrifugation at 20,000 x g for 10 minutes at 4ºC. Supernatant (10 µL) was removed, added to 300 µL Trizol, and frozen at -80ºC for future RNA-seq library preparation. RNA concentration in the remaining supernatant was quantified using RNA Qubit. Lysate (30 µg) was subjected to RNase I digestion (0.5 U RNaseI per µg RNA) at room temperature for 15 minutes with gentle agitation. After RNase digestion, 10 µL SuperasIN (Thermo) was added to each sample, and the samples were transferred to ice. To isolate ribosome protected fragments, the RNase-digested lysate was transferred to Ultra-clear 11x34 mm centrifuge tubes (Beckman Coulter) and underlaid with 0.9 mL sucrose cushion. Samples were centrifuged in a TLS-55 rotor at 51,000 rpm for 2 hours at 4°C. The supernatant was discarded and the pellet was resuspended in 300 µL TRIzol. Ribosome-protected fragments were purified from TRIzol using the Zymo Direct-zol kit. RNA was precipitated by adding 38.5 µL RNase-free water, 1.5 µL Glycoblue, 10 µL 3 M sodium acetate pH 5.5, and 150 µL isopropanol to 100 µL eluted RNA. The mixture was incubated overnight at -20°C. Samples were centrifuged for 30 minutes at 20,000 x g at 4°C. The supernatant was discarded, and the RNA pellet was resuspended in 5 µL 10 mM Tris pH 8. Five µL 2X denaturing sample loading buffer (980 µL formamide, 20 µL 500 mM EDTA, 300 µg bromophenol blue) was added to each sample, then the sample was denatured at 80°C for 90 seconds. Ribosome-protected fragments, along with control oligos66, were run on a 15% polyacrylamide gel at 200 V with 12 µL NEB miRNA marker. The gel was stained with SYBR gold in 1X TBE. Gel fragments between 17 and 34 nucleotides were excised and placed in a microfuge tube with 400 µL gel extraction buffer. Samples were frozen on dry ice for 30 minutes, then thawed overnight with gentle agitation. After overnight gel extraction, 400 µL eluate was transferred to a new microfuge tube. The RNA was precipitated by adding 1.5 µL glycoblue and 500 µL isopropanol. After overnight incubation at -20°C, the sample was centrifuged at 20,000 x g for 30 minutes at 4°C. The supernatant was discarded, and precipitated RNA was resuspended in 4 µL 10mM Tris pH 8. Samples were then dephosphorylated using T4 PNK (4 µL RNA in 10 mM Tris pH 8, 0.5 µL T4 PNK enzyme, 0.5 µL T4 PNK buffer, and 0.5 µL Superasin) at 37°C for 1 hour. Samples were then subjected to SPRI clean up: 50 µL of sample in RNase-free water was added to 90 µL RNAclean beads and 270 µL isopropanol. After washing with 85% ethanol, beads were resuspended in 7 µL RNase-free water. The supernatant was collected, and we proceeded with next-generation sequencing library preparation using the Clontech smRNA library prep kit according to the manufacturer’s instructions. Libraries were sequenced on an Illumina NovaSeq S2 with single-end 1x50 nt reads. Samples were always processed in large batches of a maximum of 24 samples to avoid sample processing biases.
RNA-seq libraries were prepared from 10 ng total RNA using the SMARTer Stranded Total RNA-seq Pico Input Mammalian V2 kit (Takara Bio) according to the manufacturer’s instructions. Samples were multiplexed with Illumina TruSeq HT barcodes and sequenced on a NextSeq 2000 with single-end 1x75 nt reads. Samples were always processed in large batches of a maximum of 24 samples to avoid sample processing biases.

treatment_protocol: On the day prior to cell harvest, neurons were silenced with TTX and APV, which antagonize sodium channels and NMDA receptors, respectively. H9 NGN2 cells were cultured in mTeSR Plus media (STEMCELL Technologies) on tissue culture plates coated with hESC-qualified matrigel (Corning). They were passaged using Dispase (1 mg/mL, Life Technologies) until ready for differentiation. A previously published protocol that combines developmental patterning and NGN2 induction was adapted to differentiate H9 NGN2 into neurons.57 At day 0, cells were treated with Accutase (StemPro Accutase, Life Technologies) and plated in single cells at 50,000 cells/cm2 in mTeSR Plus media supplemented with 10 µM Y-27632 (STEMCELL Technologies) on tissue culture plates coated with 336.67 µg/mL Growth Factor Reduced matrigel (Corning). On day 1, the medium was replaced with KSR media (Knockout DMEM medium, 15% knockout serum replacement (KOSR), 2 mM L-Glutamine, 1X MEM non-essential amino acids (MEM NEAA), 1X penicillin/streptomycin (pen/strep) and 1X 2-mercaptoethanol (all Gibco)) supplemented with 100 nM LDN193189, 2 µM XAV939 (STEMCELL Technologies), 10 µM SB431542 hydrate and 2 µg/mL doxycycline hyclate (Sigma). Day 2 media was 50% KSR media/50% NIM media supplemented with LDN/XAV/SB and 2 µg/mL doxycycline. NIM media consisted of DMEM/F-12 medium, 1X GlutaMAX, 1X MEM NEAA, 1X pen/strep, 0.16% D-glucose (Sigma) and 1X N2 supplement-B (STEMCELL Technologies). Day 3 media was NIM media supplemented with 2 µg/mL doxycycline. At day 4, cells were treated with Accutase and plated in single cells at 40,000 cells/cm2 in NB media (Neurobasal medium (without glutamine), 1X GlutaMAX, 1X MEM NEAA, 1X pen/strep and 1X N2 supplement-B) supplemented with 1X B27 without Vitamin A, 2-2.4 µg/mL mouse laminin (Gibco), 1 µM ascorbic acid, 2 µM dibutyryl cyclic-AMP (Sigma), 20 ng/mL brain-derived neurotrophic factor, 10 ng/mL glial-derived neurotrophic factor (rhBDNF and rhGDNF, Peprotech), 10 µM Y-27632 and 2 µg/mL doxycycline on the tissue culture plates coated with 336.67 µg/mL Growth Factor Reduced matrigel. The media at day 4 without Y-27632 and doxycycline is referred to as complete NB (cNB) media. On day 5 the media was replaced with cNB media. Thereafter, half of the media was replaced weekly with cNB2x, where concentrations of all the supplements to the NB media (except Y-27632) were doubled. In between each media change, media was directly supplemented with 2 µg/mL doxycycline on the third day of the week (days 8, 15 and 22). Cells were silenced on day 27 with TTX and APV, which antagonize sodium channels and NMDA receptors, respectively. Cells were collected at day 28 in 1X PBS supplemented with 1X cycloheximide after being stimulated with 55 mM KCl for 0 or 6 hours.

tag: tag

value: hESC-derived neurons


title: PRO-seq-02-SPT6AID-Veh2h-NHS1h

description: PRO-seq


title: PRO-Seq_RPB10_IAA_rep1

description:
PRO-Seq_RPB10_IAA_plus.bw
PRO-Seq_RPB10_IAA_minus.bw

value: RPB10 degron 1h mESCs


title: ChIP-seq-01-RPB1N-SPT6AID-Veh-2h

description: ChIP-seq Pol II (total)

value: Vehcle 2h


title: mRNA, ndx1-4, rep1

city: Debrecen

first: Márton

last: Miskei

address: Egyetem tér 1.

department: Department of Molecular Biotechnology and Microbiology

country: Hungary

institute: University of Debrecen

description:
single mRNA-seq ndx1-4 rep1
ndx1-4_vs_col0_diff_genes.tsv; ndx1-4_vs_col0_diff_tes.tsv

data_processing:
Reads were mapped to the A. thaliana reference transcriptome (Araport11) to quantify gene expression whilst reads were mapped to transposable elements (Araport11) themselves to estimate their expression.
For data visualization, RNA-seq reads were aligned to the TAIR10 reference genome using HISAT2 allowing for reporting spliced alignments.
We used deepTools bamCoverage to create RPKM (Reads Per Kilobase per Million) normalized bedgraph files.
Differentially expressed genes and differentially expressed transposable elements (ndx1-4 vs. Col-0 wild type) were identified by DESeq2.
Assembly: A. thaliana reference transcriptome (Araport11)
Supplementary files format and content: bigwig files are from the RPKM (Reads Per Kilobase per Million) normalized bedgraph files (2-2 replicas) of Col0 and ndx1-4 samples; tsv files (genes and transposable elements) contain the expression data (FPKM) from Col0 and ndx1-4 samples (2-2 replicas), log2 fold change, p-value (adjusted)

organism: Arabidopsis thaliana

source_name: 10-day-old seedling

growth_protocol: 10-day-old seedlings of Col-0, and ndx1-4 plants were harvested

extract_protocol:
Total RNA was purified from Col-0 and ndx1-4 seedlings using the phenol-chloroform extraction method. The quality of total RNA samples was checked by an Agilent bioanalyzer (RIN > 9).
cDNA libraries were prepared according to Illumina's TruSeq RNA Library Preparation Kit v2 protocol. NGS libraries were sequenced using an Illumina MiSeq instrument with 1x50 bp reads (Biological Research Center of Szeged, Hungarian Academy of Sciences).

value: seedling

value: ndx1-4 mutant


title: XT8698_ATAC

first: Li

last: Jiang

address: 360 Longwood Ave

department: Pediatric Oncology

institute: Dana Farber Cancer Institute

description:
snATAC-seq
barcode appendix: -8

data_processing:
Illumina bcl2fastq 1.5 is used for demultiplexing
RNA-seq sequenced reads were mapped to hg19  using hisat2(2.1.0) with parameters --met 5 -k 10 --mp 1,1 --np 1 --score-min L,0,-0.1 --secondary --no-mixed --no-softclip --no-discordant --rdg 99999999,99999999 --rfg 99999999,99999999 --no-spliced-alignment --seed 12345
RNA-seq TPM were calculated using RSEM (v1.3.0) with parameters --estimate-rspd --paired-end -sam
ATAC-seq data were analyzed using cellranger-ATAC (V1.1.0) count and aggr, using default parameterss
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include TPM values for all cells and cells past QC (for fresh and frozen samples separately). Tab-delimited text files for metadata of cells past QC

source_name: Human H3-K27M DMG

extract_protocol:
Smart-seq2 protocol, Chromium Next GEM Single Cell ATAC Protocol
Illumina, Nextera XT Library Prep kit,  Chromium Next GEM Single Cell ATAC & Library Gel Bead Kit v1.1 kit

tag: tissue type

value: Human H3-K27M DMG

tag: age at diagnosis

value: male

tag: location

value: lower brainstem


title: mRNA, ndx1-4, rep2

description:
single mRNA-seq ndx1-4 rep2
ndx1-4_vs_col0_diff_genes.tsv; ndx1-4_vs_col0_diff_tes.tsv


title: PRO-Seq_RPB10_IAA_rep2


title: mRNA, col0, rep2

description:
single mRNA-seq Col0 rep2
ndx1-4_vs_col0_diff_genes.tsv; ndx1-4_vs_col0_diff_tes.tsv

value: wild type


title: PRO-Seq_RPB10_Untreated_rep1

description:
PRO-Seq_RPB10_Untreated_plus.bw
PRO-Seq_RPB10_Untreated_minus.bw

value: RPB10 untreated mESCs


title: mRNA, col0, rep1

description:
single mRNA-seq Col0 rep1
ndx1-4_vs_col0_diff_genes.tsv; ndx1-4_vs_col0_diff_tes.tsv


title: Ribo_6h_KCl_rep2

description: Ribosome-associated RNA


title: Ribo_6h_KCl_rep1

description: Ribosome-associated RNA


title: XT8698

description: frozen single nucleus RNA-seq

molecule: polyA RNA


title: PRO-Seq_RPB9_IAA_rep2

description:
PRO-Seq_RPB9_IAA_plus.bw
PRO-Seq_RPB9_IAA_minus.bw

value: RPB9 degron 1h mESCs


title: PRO-Seq_RPB9_IAA_rep1


title: S1D2E3H7

description: frozen single nucleus RNA-seq

value: female

value: thalamus


title: Ribo_0h_KCl_rep2

description: Ribosome-associated RNA


title: Ribo_0h_KCl_rep3

description: Ribosome-associated RNA


title: MGH6666410

description: frozen single nucleus RNA-seq

value: pons


title: MKN45-64839-2

city: Lanzhou

first: Fan

last: Zhang

state: Gansu Province

address: 82 Cuiyingmen

department: Department of Oncology Surgery

institute: Lanzhou university

description: MKN45 cells, LV-ZNF165

data_processing:
Hisat2（version:2.0.4）
Stringtie（version:1.3.0）
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

extract_protocol:
Total RNA was extracted using RNAiso Plus Total RNA extraction reagent (TAKARA, Cat#9109) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
RNA libraries for RNA-seq were prepared using Qubit 2.0 Fluorometer following manufacturer's protocols.

value: gastric cancer cell

value: ZNF165 Overexpression


title: MKN45-64839-3

description: MKN45 cells, LV-ZNF165


title: R1E1E3G3

description: frozen single nucleus RNA-seq


title: PRO-Seq_RPB9_Untreated_rep2

description:
PRO-Seq_RPB9_Untreated_plus.bw
PRO-Seq_RPB9_Untreated_minus.bw

value: mouse embryonic stem cells (V6.5)

value: RPB9 untreated mESCs


title: PRO-Seq_RPB10_Untreated_rep2


title: Ribo_0h_KCl_rep1

description: Ribosome-associated RNA

value: hESC-derived neurons


title: MKN45-64839-1

description: MKN45 cells, LV-ZNF165


title: MUV82_ATAC

description:
snATAC-seq
barcode appendix:  -5


title: D538G_4_102

city: London

first: Simak

last: Ali

address: Du Cane Road

department: Department of Surgery and Cancer

country: United Kingdom

institute: Imperial College London

data_processing:
Bcbio 1.1.1 RNA-seq pipeline was used for quality control using Samtools, QualiMap and FastQC
Reads were aligned using Hisat2 2.1.0
Reads were counted using Dexseq and Salmon 0.11.3
Reads were normalised using DESeq2
Genome_build: hg38
Supplementary_files_format_and_content: csv file containing raw read counts for each sample

source_name: MCF7-Luc cells

growth_protocol: Cell lines were routinely cultured in DMEM containing 10% FCS

extract_protocol:
Total RNA was extracted for six biological replicates, using QIAGEN RNeasy Mini Preparation Kit
Samples were sent to Beijing Genomics Insitute for quality assessment, library preparation using NEBNext® Ultra™ RNA Library Prep Kit and RNA-sequencing

value: MCF7-Luc cells

tag: mutation status (esr1)


title: MKN45-3

description: MKN45 cells, LV-Control

value: WT


title: MKN45-2

description: MKN45 cells, LV-Control


title: MUV82

description: frozen single nucleus RNA-seq


title: PRO-Seq_RPB8_IAA_rep2

description:
PRO-Seq_RPB8_IAA_plus.bw
PRO-Seq_RPB8_IAA_minus.bw

value: RPB8 degron 1h mESCs


title: SUDIPG67

description: frozen single nucleus RNA-seq

value: Human H3-K27M DMG


title: PRO-Seq_RPB8_IAA_rep1


title: D538G_4_101


title: D538G_4_098


title: MKN45-1

description: MKN45 cells, LV-Control


title: D538G_4_099


title: D538G_4_100


title: PRO-Seq_RPB8_Untreated_rep2

description:
PRO-Seq_RPB8_Untreated_plus.bw
PRO-Seq_RPB8_Untreated_minus.bw

value: RPB8 untreated mESCs


title: XT9824

description: frozen single nucleus RNA-seq

value: male


title: MUV32

description: fresh single whole cell RNA-seq


title: PRO-Seq_RPB8_Untreated_rep1


title: MKN28-97009-3

description: MKN28 cells, LV-ZNF167-siRNA

value: ZNF165 knockdown


title: MKN28-97009-2

description: MKN28 cells, LV-ZNF166-siRNA


title: D538G_4_097


title: MUV31_ATAC

description:
snATAC-seq
barcode appendix: -3


title: PRO-Seq_RPB7_IAA_rep2

description:
PRO-Seq_RPB7_IAA_plus.bw
PRO-Seq_RPB7_IAA_minus.bw

value: RPB7 degron 1h mESCs


title: MKN28-97009-1

description: MKN28 cells, LV-ZNF165-siRNA


title: D538G_3_096


title: PRO-Seq_RPB7_IAA_rep1


title: PRO-Seq_RPB9_Untreated_rep1


title: MUV31

description: fresh single whole cell RNA-seq


title: D538G_3_095


title: MKN28-3

description: MKN28 cells, LV-Control


title: MUV91

description: fresh single whole cell RNA-seq


title: PRO-Seq_RPB7_Untreated_rep2

description:
PRO-Seq_RPB7_Untreated_plus.bw
PRO-Seq_RPB7_Untreated_minus.bw

value: RPB7 untreated mESCs


title: D538G_3_094


title: SUDIPG65

description: frozen single nucleus RNA-seq

value: female


title: D538G_3_093


title: MUV54

description: fresh single whole cell RNA-seq


title: D538G_3_092


title: R28P-30B_heavy_polysome_N2

city: Montréal

first: Frédérick

last: Mallette

state: QC

address: 5415 boulevard de L'Assomption

department: Médecine

country: Canada

institute: Université de Montréal

description: RNA from heavy polysome fractions (Poly2+Poly3), polyA enriched, fragmented (200nt)

data_processing:
TrimmomaticPE: Started with arguments: -phred33 R1.fastq.gz R2.fastq.gz R1.trimmed.paired.fastq.gz R1.trimmed.unpaired.fastq.gz R2.trimmed.paired.fastq.gz R2.trimmed.unpaired.fastq.gz ILLUMINACLIP:Adapters-PE_NextSeq.fa:2:12:10:8:true TRAILING:30
STAR --runMode genomeGenerate --genomeDir genome/ --genomeFastaFiles Saccharomyces_cerevisiae.R64-1-1.dna.chromosome.all.fa --sjdbGTFfile Saccharomyces_cerevisiae.R64-1-1.90.gtf --sjdbOverhang 75
STAR --runMode alignReads --genomeDir genome/ --readsFilesIn R1.trimmed.paired.fastq.gz R2.trimmed.paired.fastq.gz --readsFilesCommand zcat --outReadsUnmapped Fastx --outFilterType BySJout --outStd Log --outSAMunmapped None --outSAMtype BAM SortedByCoordinate
Rsubread featureCounts(Aligned.sortedByCoord.out.bam, genome=Saccharomyces_cerevisiae.R64-1-1.dna.chromosome.all.fa, annot.ext=Saccharomyces_cerevisiae.R64-1-1.90.gtf, isGTFAnnotationFile=TRUE, GTF.featureType="exon", GTF.attrType="gene_id",countChimericFragments=FALSE, largestOverlap=FALSE, isPairedEnd=TRUE, useMetaFeatures=TRUE, requireBothEndsMapped=TRUE, strandSpecific=2, minOverlap=10, autosort=TRUE, allowMultiOverlap=FALSE, reportReads=TRUE, juncCounts=FALSE,fraction=FALSE, countMultiMappingReads=FALSE)
Assembly: Saccharomyces_cerevisiae.R64-1-1
Supplementary files format and content: comma-separated-value (csv) files holding the name of every gene and their abundance value for each sample(CPM or TPM)

organism: Saccharomyces cerevisiae

growth_protocol: Cells grown in Yc media at 30°C

extract_protocol:
RNA was extracted from cultures growing in exponential phase. Total RNA was extracted from the culture. Light and heavy fractions RNA were purified after polysome fractionation
RNA libraries were prepared using NEBNExt Poly(A) mRNA Isolation Module and NEBNExt Ultra II Directional RNA Library Prep Kit

tag: strain

value: BY background

tag: growth protocol

value: grown to log phase in synthetic minimal media


title: SUDIPG31

description: frozen single nucleus RNA-seq

value: pons


title: R28P-30B_heavy_polysome_N1


title: D538G_3_091

tag: cell line


title: PRO-Seq_RPB6_IAA_rep1

description:
PRO-Seq_RPB6_IAA_plus.bw
PRO-Seq_RPB6_IAA_minus.bw

tag: treatment

value: RPB6 degron 1h mESCs


title: PRO-Seq_RPB6_IAA_rep2

value: mouse embryonic stem cells (V6.5)


title: SUDIPG45

description: frozen single nucleus RNA-seq


title: PRO-Seq_RPB6_Untreated_rep2

description:
PRO-Seq_RPB6_Untreated_plus.bw
PRO-Seq_RPB6_Untreated_minus.bw

value: RPB6 untreated mESCs


title: D538G_2_090


title: PRO-Seq_RPB6_Untreated_rep1


title: SUDIPG30

description: frozen single nucleus RNA-seq


title: R28P-30B_light_polysome_N2

description: RNA from light polysome fractions (40S+60S+80S), polyA enriched, fragmented (200nt)


title: D538G_2_089


title: ICGCGBM96

description: frozen single nucleus RNA-seq

value: thalamus


title: R28P-30B_light_polysome_N1


title: R28P-30B_total_extract_N2

description: Total RNA, polyA enriched, fragmented (200nt)


title: PRO-Seq_RPB7_Untreated_rep1


title: D538G_2_088

value: MCF7-Luc cells


title: PRO-Seq_RPB5_IAA_rep2

description:
PRO-Seq_RPB5_IAA_plus.bw
PRO-Seq_RPB5_IAA_minus.bw

value: RPB5 degron 1h mESCs


title: PRO-Seq_RPB5_IAA_rep1


title: ICGCGBM60

description: frozen single nucleus RNA-seq


title: D538G_2_087


title: MKN28-2

description: MKN28 cells, LV-Control

value: gastric cancer cell

value: WT


title: PRO-Seq_RPB5_Untreated_rep2

description:
PRO-Seq_RPB5_Untreated_plus.bw
PRO-Seq_RPB5_Untreated_minus.bw

value: RPB5 untreated mESCs


title: MKN28-1

description: MKN28 cells, LV-Control


title: pTG4

description: frozen single nucleus RNA-seq

value: Human H3-K27M DMG


title: R28P-30B_total_extract_N1


title: SUDIPGIII

description: frozen single nucleus RNA-seq

value: male


title: PRO-Seq_RPB5_Untreated_rep1


title: R28P-30A_heavy polysome_N2


title: D538G_2_085


title: MUV86_ATAC

description:
snATAC-seq
barcode appendix: -6


title: D538G_1_084


title: R28P-30A_light_polysome_N2


title: MUV86

description: fresh single whole cell RNA-seq


title: D538G_1_083


title: pSCG1

description: frozen single nucleus RNA-seq

value: spinal cord


title: PRO-Seq_RPB4_Untreated_rep2

description:
PRO-Seq_RPB4_Untreated_plus.bw
PRO-Seq_RPB4_Untreated_minus.bw

value: RPB4 untreated mESCs


title: D538G_1_082


title: pTG5

description: frozen single nucleus RNA-seq

value: female


title: D538G_1_081


title: R28P-30A_total_extract_N2

value: BY background


title: R28P-30A_total_extract_N1

value: grown to log phase in synthetic minimal media


title: D538G_2_086


title: MUV17

description: fresh single whole cell RNA-seq

value: pons


title: WT_heavy_polysome_N2


title: D538G_1_080


title: PRO-Seq_RPB3_IAA_rep1

description:
PRO-Seq_RPB3_IAA_plus.bw
PRO-Seq_RPB3_IAA_minus.bw

value: RPB3 degron 1h mESCs


title: PRO-Seq_RPB3_IAA_rep2

value: mouse embryonic stem cells (V6.5)


title: R28P-30A_heavy_polysome_N1


title: SUDIPG52

description: frozen single nucleus RNA-seq


title: PRO-Seq_RPB4_IAA_rep2

description:
PRO-Seq_RPB4_IAA_plus.bw
PRO-Seq_RPB4_IAA_minus.bw

value: RPB4 degron 1h mESCs


title: PRO-Seq_RPB3_Untreated_rep2

description:
PRO-Seq_RPB3_Untreated_plus.bw
PRO-Seq_RPB3_Untreated_minus.bw

value: RPB3 untreated mESCs


title: WT_heavy_polysome_N1


title: Y537S_3_012

value: MCF7-Luc cells


title: D538G_1_079


title: R28P-30A_light_polysome_N1


title: PRO-Seq_RPB3_Untreated_rep1


title: PRO-Seq_RPB4_IAA_rep1


title: SUDIPG54

description: frozen single nucleus RNA-seq


title: Y537S_3_011


title: MUV77

description: fresh single whole cell RNA-seq


title: WT_light_polysome_N2


title: PRO-Seq_RPB4_Untreated_rep1


title: WT_light_polysome_N1


title: Y537S_3_010


title: WT_total_extract_N2


title: PRO-Seq_RPB2_IAA_rep1

description:
PRO-Seq_RPB2_IAA_plus.bw
PRO-Seq_RPB2_IAA_minus.bw

value: RPB2 degron 1h mESCs


title: BT1126

description: fresh single whole cell RNA-seq


title: Y537S_3_009


title: MUV10

description: fresh single whole cell RNA-seq

value: thalamus


title: WT_total_extract_N1


title: PRO-Seq_RPB2_Untreated_rep2

description:
PRO-Seq_RPB2_Untreated_plus.bw
PRO-Seq_RPB2_Untreated_minus.bw

value: RPB2 untreated mESCs


title: Y537S_3_008


title: BS, ndx1-4, rep2

description:
paired-end BS-seq ndx1-4 rep2
ndx1-4_vs_col0_DMR.tsv
ndx1-4_vs_col0.CG.DMR.bed; ndx1-4_vs_col0.CHG.DMR.bed; ndx1-4_vs_col0.CHH.DMR.bed

data_processing:
The Bismark software86 was used to align the bisulfite-treated reads to the TAIR10 reference genome.
Methylation levels were determined for CpG, CHH, and CHG sequence contexts as percentage of methylated cytosines in their contexts and integrated with JBrowse.
Differentially methylated regions (DMRs) were identified by the DSS-single (DSS) pipeline considering the variance among biological replicates.
To compare the DNA methylome changes of ndx1-4 plants to a large collection of DNA methylation mutants functioning in different pathways, we used the hcDMR pipeline. High-confidence DMRs were identified by comparing the methylation changes of each mutant to the methylation levels of 54 control libraries.
Assembly: A. thaliana reference genome (TAIR10; NCBI; Ecotype: Columbia-0)
Supplementary files format and content: The bigwig files are from the RPKM (Reads Per Kilobase per Million) normalized bedgraph files (2-2 replicas) of Col0 and ndx1-4 samples; The tsv files contain differentially methylated regions (DMR) from Col0 and ndx1-4 samples; The bed files contain the different DMRs (CG, CHG, CHH)

source_name: 14-day-old seedling

growth_protocol: Genomic DNA was prepared by standard phenol-chloroform extraction from 14-old seedlings (Col-0 and ndx1-4).

extract_protocol: 1ng/l of PE150 library was prepared and sequenced on an Illumina NovaSeq 6000 instrument resulting in 15-17 million raw reads.

treatment_protocol: DNA samples were fragmented into 200-400bp using Covaris S220 and end-repaired, dA-tailed and ligated to sequencing adaptors containing only methylated cytosines. Then the DNA fragments were sodium-bisulfite treated with EZ DNA Methylation Gold Kit (Zymo Research) after which cytosines without methylation changed to U (after PCR amplification to T), while cytosines with methylation remained unchanged.

tag: tissue

value: seedling

value: ndx1-4 mutant


title: PRO-Seq_RPB2_Untreated_rep1

source_name: mouse embryonic stem cells (V6.5)


title: PRO-Seq_RPB1_IAA_rep2

description:
PRO-Seq_RPB1_IAA_plus.bw
PRO-Seq_RPB1_IAA_minus.bw

value: RPB1 degron 1h mESCs


title: Y537S_3_007


title: SUDIPG50Aut

description: frozen single nucleus RNA-seq

value: Human H3-K27M DMG

tag: gender


title: BS, ndx1-4, rep1

description:
paired-end BS-seq ndx1-4 rep1
ndx1-4_vs_col0_DMR.tsv
ndx1-4_vs_col0.CG.DMR.bed; ndx1-4_vs_col0.CHG.DMR.bed; ndx1-4_vs_col0.CHH.DMR.bed


title: SUDIPG50Bio

description: frozen single nucleus RNA-seq


title: ICGCGBM27

description: frozen single nucleus RNA-seq

value: male


title: Y537S_2_024


title: PRO-Seq_RPB1_IAA_rep1


title: Y537S_2_023


title: PRO-Seq_RPB2_IAA_rep2


title: MUV16_ATAC

description:
snATAC-seq
barcode appendix: -2


title: BT1462

description: fresh single whole cell RNA-seq

value: female

value: spinal cord


title: Y537S_2_022


title: PRO-Seq_RPB1_Untreated_rep2

description:
PRO-Seq_RPB1_Untreated_plus.bw
PRO-Seq_RPB1_Untreated_minus.bw

value: RPB1 untreated mESCs


title: BS, col0, rep1

description:
paired-end BS-seq Col0 rep1
ndx1-4_vs_col0_DMR.tsv
ndx1-4_vs_col0.CG.DMR.bed; ndx1-4_vs_col0.CHG.DMR.bed; ndx1-4_vs_col0.CHH.DMR.bed

value: wild type


title: MUV16

description: frozen single nucleus RNA-seq


title: BS, col0, rep2

description:
paired-end BS-seq Col0 rep2
ndx1-4_vs_col0_DMR.tsv
ndx1-4_vs_col0.CG.DMR.bed; ndx1-4_vs_col0.CHG.DMR.bed; ndx1-4_vs_col0.CHH.DMR.bed


title: PRO-Seq_RPB1_Untreated_rep1


title: midbrain aars1ENU-/-

city: Chongqing

first: Binbin

last: Jin

address: NO.2 Rd Tiansheng, Beibei District

department: Institute of Developmental Biology and Regenerative Medicine, Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education

institute: Southwest University

description: zebrafish midbrain tissue

data_processing:
The scRNA-seq data were aligned to the zebrafish reference genome (Danio rerio, GRCz11) by cellranger-5.0.0. The resulting matrix was inputted into Seurat v3 for quality control, filtration, dimensionality reduction, clustering analysis and differential expressed genes analysis
Genome_build: GRCz11
Supplementary_files_format_and_content: Cell Ranger output files (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz)

organism: Danio rerio

source_name: midbrain

extract_protocol: The 10x RNA-seq libraries were constructed using Chromium single cell gene expression platform and Chromium Chromium Single Cell 3' Reagent v3 Kits according to the manufacture's protocol.

tag: developmental stage

value: 2 dpf

value: midbrain

value: aars1ENU-/- mutant


title: midbrain sibling

description: zebrafish midbrain tissue

tag: genotype

value: sibling


title: PRO-Seq_WT_IAA_rep2

description:
PRO-Seq_WT_IAA_plus.bw
PRO-Seq_WT_IAA_minus.bw

value: mouse embryonic stem cells (V6.5)

value: WT degron 1h mESCs


title: Y537S_2_021

value: MCF7-Luc cells


title: BT869

description: fresh single whole cell RNA-seq

value: pons


title: Y537S_2_020


title: MUV83

description: fresh single whole cell RNA-seq

value: thalamus


title: PRO-Seq_WT_IAA_rep1


title: PRO-Seq_WT_Untreated_rep2

description:
PRO-Seq_WT_Untreated_plus.bw
PRO-Seq_WT_Untreated_minus.bw

value: WT untreated mESCs


title: Y537S_2_019


title: d7_2

first: Rui-Ting

last: Li

state: Beijing

address: No.20 Dongda Street, Fengtai District

department: State Key Laboratory of Pathogen and Biosecurity

institute: Beijing Institute of Microbiology and Epidemiology

description: 7 dpi

data_processing:
After sequencing, perl script was used to filter the original data (Raw Data) to clean reads by removing contaminated reads for adapters and low-quality reads.
Clean reads were aligned to the cavia_porcellus genome using Hisat2 v2.1.0. The number of reads mapped to each gene in each sample was counted by HTSeq v0.6.0 and FPKM was then calculated  to estimate the expression level of genes in each sample.
Assembly: Cavpor3.0
Supplementary files format and content: tab-delimited text files include FPKM values for each sample

organism: Cavia porcellus

source_name: lung

extract_protocol:
Total RNA from lung  were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA)  and DNase I (NEB, USA) treated, respectively.
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations

value: lung

value: ZIKV infected


title: d7_3

description: 7 dpi


title: SUDIPG55_ATAC

description:
snATAC-seq
barcode appendix: -7

value: Human H3-K27M DMG


title: Y537S_1_018


title: BT1179

description: frozen single nucleus RNA-seq

value: female


title: SUDIPG55

description: frozen single nucleus RNA-seq


title: RNA-Seq_RPB6_IAA_1h_rep2

data_processing:
Cutadapt V1.18 (Martin, 2011) was used to remove the adaptors (TELP libraries adaptor) and low-quality reads from the paired-end sequencing reads, then the filtered reads were mapped to the mm10 genome with bowtie2 (--very-sensitive-local) (Langmead and Salzberg, 2012).
In order to obtain the more reliable nascent RNA signals, the duplicate, discordant and multi-mapping reads were removed, rRNA reads were also removed with split_bam.py from RseQC V 2.6.6 (Wang et al., 2012).
The remaining reads were adapter trimmed and aligned against the concatenated genome of mm10 and dm6 using the STAR aligner (Dobin et al., 2013) with default parameters, and then subsequently separated into the mouse and fly bins. Finally, non-redundant reads were determined by samtools
To display the information of the plus and minus chains at the same time, bamCoverage (Ramirez et al., 2014) was used to make the tracks for plus and minus chains separately with parameters –filterRNAstrand forward/reverse and –scaleFactor.
The uniquely aligned reads were counted over the Gencode annotated genes (M23) using featureCounts, and the output files from two replicates of each group were subsequently analyzed with a custom R script utilizing the DESeq2 package (Love et al., 2014), which incorporates spike-in calibration for data normalization and differential gene expression analysis.
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq raw read counts per sample per replicate in tab-delimited text format.

growth_protocol: For RNA-seq, wild-type, Pol II subunit degron cells and piRPB3 degron cells were cultured in DMEM-KO (Thermo Fisher, Cat#10829018) supplemented with 15% FBS (Gibco, Cat#10099141C), 1× penicillin/ streptomycin (Gibco, Cat#15140122), 1 × L-glutamine (Gibco, Cat#25030081), 1 × non-essential amino acids (Gibco, Cat#11140050), 1 × nucleoside (Millipore, Cat#ES-008-D), 3 μM CHIR99021 (Selleck, Cat#S1263), 0.5 μM PD0325901 (Selleck, Cat#S1036), 143 uM 2-mercaptoethanol (Thermo Fisher, Cat#M3148) and maintained at 37℃ cell incubator with 5% CO2. The Schneider 2(S2) cells were cultured in complete Schneider’s Drosophila Medium (Invitrogen, Cat#21720024) supplemented with 10% FBS (CellMax, Cat#SA311.02) at room temperature.

extract_protocol:
Total RNA were extracted according to the manufacturer’s instructions.
Doxycycline and IAA 12h treatment samples were delivered to Novogene Company. The total RNA's concentration, purity, and integrity were examined and followed by PolyA RNA library generation.

treatment_protocol: Cells were treated with 2 μg/ml Doxycycline for 12 h to induce OsTir1 expression, then treated with or without 500 uM IAA for 12 h to induce target protein degradation. For RNA extraction, about 106 mES cells and 10% S2 cells were suspended in a 0.5 ml TRIzol reagent.

value: RPB6 degron 1h mESCs


title: d3_3

description: 3 dpi


title: Y537S_1_016


title: PRO-Seq_WT_Untreated_rep1

data_processing:
Library strategy: PRO-Seq
PRO-seq raw data were processed using a custom pipeline that builds on published workflows with minor modifications. Briefly, ribosomal reads were first removed by mapping to one copy of the mouse rDNA sequence. After adapter trimming and quality control with cutadapt and RseQC separately, clean reads were then aligned to a concatenated genome comprised of mouse mm10 and fly dm6 assemblies using bowtie2 with options (-very-sensitive-local) as described in the ChIP-seq section.
Only reads that were properly paired and uniquely aligned were retained for subsequent processing. Reads that mapped to the mouse genome were normalized to total reads originating from Drosophila spike-in cells, and the normalization factor was calculated as previously described (Jiang et al., 2020).
Plus- and minus-strand were assigned to uniquely mapped reads using SAMtools, and the final BAM files were converted to bigwig, separated by strand, and normalized to spike-in controls using bamCoverage from deeptools. Finally, bigWig score tracks for visualization prepared for different biological replicates but for the same treatment were merged to an averaged signal using the mean operator from the WiggleTools (v1.2) package (Zerbino, Johnson, et al. 2014).
Then raw read counts were calculated per gene, in a strand-specific manner, based on annotations described in the Gene list definition section below, using featureCounts (Liao et al., 2014).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files

extract_protocol:
1-Biotin run-on was applied according to the protocol of Dig et al. (Dig et al., 2016). Total RNA was extracted using TRIzol reagent and fragmented by base hydrolysis. The nascent RNA labeled by Biotin were enriched with streptavidin beads and extracted using TRIzol reagent again.
The enriched RNA was reverse transcribed into first-strand DNA using SuperScript IV reverse transcriptase kit (Invitrogen, 18090010) with N9 random primer. And RNA was eliminated by adding 2μl 1M NaOH and heating at 98℃ for 20min. DNA was purified with Phenol/Chloroform and precipitated with Ethanol. Finally, the DNA samples were subjected to TELP library construction (Peng et al., 2015). 200bp-400bp DNA libraries were gel-selected, purified, and subjected to HiSeq X-ten PE150 sequencing.
TELP libraries


title: d3_2

description: 3 dpi


title: RNA-Seq_RPB6_IAA_0h_rep2

value: RPB6 degron 0h mESCs


title: Y537S_1_015


title: AAA07653

description: frozen single nucleus RNA-seq

value: male


title: BT836

description: fresh single whole cell RNA-seq


title: Y537S_1_014


title: d1_3

description: 1 dpi


title: MUV87

description: fresh single whole cell RNA-seq


title: RNA-Seq_RPB6_IAA_0h_rep1


title: Y537S_1_013


title: d3_1

description: 3 dpi


title: d7_1

description: 7 dpi


title: MUV35_ATAC

description:
snATAC-seq
barcode appendix: -4


title: Y537N_3_036


title: d1_1

description: 1 dpi


title: RNA-Seq_RPB3_IAA_1h_rep2

value: RPB3 degron 1h mESCs


title: Y537S_1_017


title: MUV35

description: fresh single whole cell RNA-seq


title: RNA-Seq_RPB3_IAA_1h_rep1


title: Y537N_3_035


title: RNA-Seq_RPB6_IAA_1h_rep1


title: MUV1_ATAC

description:
snATAC-seq
barcode appendix: -1


title: RNA-Seq_RPB3_IAA_0h_rep2

value: RPB3 degron 0h mESCs


title: d0_2

description: 0 dpi

value: mock


title: RNA-Seq_RPB3_IAA_0h_rep1


title: MUV1

description: fresh single whole cell RNA-seq


title: Y537N_3_034


title: MUV78

description: fresh single whole cell RNA-seq


title: Y537N_3_032


title: Y537N_3_033


title: RNA-Seq_RPB1_IAA_1h_rep2

value: RPB1 degron 1h mESCs


title: CBR30 Coarctation

city: East Lansing

first: Genaro

middle: Andres

last: Contreras

state: MI

address: 736 Wilson Road Room D202, Michigan State University

department: Large Animal Clinical Sciences

institute: Michigan State University

data_processing:
For bulk mRNA sequencing, samples were transcriptomic-sequenced via Illumina platform by Novogene. After quality control, the paired-end clean reads were mapped onto reference genome using STAR (v2.5). HTSeq (v0.6.1) was used to count the read numbers of each gene. The gene read counts matrix was further fed into DESeq2 (v1.24.0) to identify the differentially expressed genes (DEGs). These DEGs were further analyzed based on the Gene Ontology database using David (v6.8) (8), g.Profiler (2019 update) (23).
Assembly: Rattus.norvegicus (v1.3.1)

organism: Rattus norvegicus

source_name: Brown Adipose Tissue

extract_protocol: RNA was extracted from thoracic aortic PVAT dissected from above and below the coarctation using the Quick RNA MiniPrep kit (Cat. No. R1054; Zymo Research, Irving, CA) that includes a DNase step to remove genomic DNA. Purity, concentration, and integrity of mRNA were evaluated using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). All samples had a 260:280 nm ratio between 1.9 and 2.1 and RNA integrity number ≥ 7.5. Samples were sent to Novogene Corporation Inc. (Sacramento, CA) for analysis and reporting of RNA-seq quantification.

treatment_protocol: Coarctation of throacic aorta: The descending thoracic aorta was exposed through an intercostal incision between the fourth and fifth ribs. An 18-gauge blunt tip needle with the tip bent 90° was placed on the aorta. A uniform degree of constriction around the descending thoracic aorta was produced by tying a 5-0 surgical silk ligature tightly around the aorta and blunt tip needle. The blunt tip needle was then withdrawn from the ligature and the chest incision was closed. Sham-operated control animals underwent the same procedure, but the ligature on the aorta was not tightened.

value: Coarctation

value: Spargue Dawley

value: SD/Rij

value: Male

value: 10-15 weeks

tag: water

value: Aquavive water (Innovive)

value: Chow diet

value: Envigo Teklad 22/5 #8640

value: 15 weeks


title: RNA-Seq_RPB1_IAA_1h_rep1

value: mouse embryonic stem cells (V6.5)


title: CBE30 Coarctation

source_name: Below Thoracic Perivascular Fat


title: Y537N_3_031

value: MCF7-Luc cells


title: MUV5

description: fresh single whole cell RNA-seq

value: Human H3-K27M DMG

value: pons


title: d1_2

description: 1 dpi

value: lung

value: ZIKV infected


title: RNA-Seq_RPB1_IAA_0h_rep2

value: RPB1 degron 0h mESCs


title: Y537N_2_030


title: Y537N_2_029


title: RNA-Seq_RPB1_IAA_0h_rep1


title: CAB30 Coarctation

source_name: Above Thoracic Perivascular Fat


title: d0_3

description: 0 dpi

value: mock


title: RNA-Seq_RPB12_IAA_rep2

value: RPB12 degron 12h mESCs


title: RNA_6h_KCl_rep3

description: total RNA

data_processing:
In an effort to capture the most complete picture of translation, including the potential translation of brain-specific lncRNAs, RNA-seq and Ribo-seq reads were mapped to the lncRNA knowledge base (lncRNAKB) annotation.68 This annotation includes experimental evidence of lncRNA expression across 31 solid human normal tissues, including the brain, providing a comprehensive resource of transcripts and transcript isoforms in the human brain. Sequencing reads were aligned using Hisat2 (version 2.1.0) to the H. sapiens genome (GRCh30) and transcriptome (lncRNAKB). Alignments and analysis were performed on the Orchestra2 high performance computing cluster through Harvard Medical School. Aligned bam files were sorted using Picard Tools (version 2.8.0), stranded bedGraphs were generated using STAR, and reads were quantified over annotated exons using HTSeq-count (version 0.9.1).
Sequencing adapters were removed using Cutadapt (version 1.14), trimmed fastq files were aligned to hg38 ribosomal RNA sequences, and unaligned reads were mapped to the hg38 genome and lncRNAKB transcriptome68 using STAR (version 2.7.3a) with standard settings and the following modified parameters: --clip5pNbases 3, --seedSearchStartLmax 15, --outSJfilterOverhangMin 30 8 8 8, --outFilterScoreMin 0, -outFilterScoreMinOverLread 0.66, -outFilterMatchNmin 0, -outFilterMatchNminOverLread 0.66, --outSAMtype BAM Unsorted. Aligned bam files were sorted using Picard Tools (version 2.8.0) and stranded bedGraphs were generated using STAR. The RibORF pipeline was run on each sample individually using standard parameters. Due to template switching during library preparation, reads contained three untemplated bases at the 3’ end that were not included in the alignment but added to the length of each read. Therefore, reads 30-33 nt in length (corresponding to RNA fragments 27-30 nt) were analyzed for three-nucleotide periodicity within known protein-coding ORFs (RefSeq). For each sample we selected only the read lengths for which at least 50% of the reads matched the primary ORF of known protein-coding genes in a meta-gene analysis. Samples with fewer than two read lengths passing filtering were removed from further analysis. Read lengths were offset-corrected and RibORF was used to predict ORFs with a minimum length of 8 AA and translation probability >0.7. After running the RibORF pipeline on each brain sample individually, information from RibORF output files was used to generate GTF and BED files for all ORFs identified in each sample. ORFs with lengths of zero and ORFs annotated as non-coding despite being detected in protein coding genes were eliminated. Using Bedtools version 2.27.1 and the GRCh38 primary assembly human genome file, DNA sequences were associated with each exon of each remaining ORF. ORFs that did not end in stop codon sequences (“TGA”,”TAA”,”TAG”) were eliminated. Using the R library micropan version 2.1, DNA sequences for each complete ORF were translated into protein sequences. Of note, ORFs with start codons “GTG” or “TTG” are translated with a Methionine as the initial amino acid despite these sequences not typically encoding methionine in other positions in a protein-coding DNA sequence, per existing literature on non-canonical start codon usage in translation. Finally, all remaining ORF information was collapsed into one table, and duplicate ORFs, defined as ORFs in the same genomic position with identical protein sequences, were eliminated. When eliminating duplicates, ORFs identified of the most common ORF type identified by RibORF were conserved, according to the following order of priority, from highest to lowest: canonical, truncation, extension, overlap, uORF, internal, external, polycistronic, readthrough, and non-coding ORFs. ORFs annotated as type “seqerror” were eliminated. After combining ORF outputs from all samples, ORFs that were only detected in one sample were eliminated. After the removal of singleton ORFs, duplicate ORFs were once again eliminated according to the same priority scheme, leaving only one entry for each ORF that was detected in at least two samples in the dataset. The LncRNAKB annotation was used to assign a specific ORF type to each ORF. In order to be designated a sORF, an ORF had to be 100 amino acids or less in length, and not fully overlap in-frame with a canonical protein-coding ORF.
Genome_build: hg38

value: hESC-derived neurons


title: RNA_6h_KCl_rep2

description: total RNA


title: CBR29 Coarctation

value: Coarctation

value: Spargue Dawley

value: SD/Rij


title: Y537N_2_027


title: Y537N_2_028


title: RNA-Seq_RPB12_IAA_rep1


title: RNA_0h_KCl_rep3

description: total RNA


title: RNA_6h_KCl_rep1

description: total RNA


title: CBE29 Coarctation

value: Male

value: 10-15 weeks

value: Aquavive water (Innovive)


title: d0_1

description: 0 dpi


title: Y537N_2_026


title: RNA-Seq_RPB12_Untreated_rep1

value: RPB12 untreated mESCs


title: SBE22 Sham

value: Sham

value: Chow diet

value: Envigo Teklad 22/5 #8640

value: 15 weeks


title: Y537N_2_025

value: MCF7-Luc cells


title: ABCB5-positive LSCs from donor 3

first: Natasha

last: Frank

address: 77 Avenue Louis Pasteur

department: Medicine

institute: Brigham and Women's Hospital

data_processing:
The reads were pre-processed with Cell Ranger v2.1.0, which demultiplexed cells from different samples and quantified transcript counts per putative cell (3,768 cells total).
Quantification was performed using the STAR aligner against the hg19 transcriptome. Cell Ranger aggr was used to combine the three libraries, normalizing based on mapped sequencing depth.
Genome_build: hg19
Supplementary_files_format_and_content: Processed data consists of TSV files of barcodes and genes corresponding to the sparse matrix MTX file

source_name: ABCB5-positive LSCs

extract_protocol:
Human donor whole globes were obtained from eye banks. Cornea was dissected from the other parts of the eyes, the central cornea was separated by an 8mm disposable biopsy punch and the corneal endothelium was removed mechanically. After 1 h incubation with PluriSTEM Dispase II Solution at 37 °C, limbal/central corneal epithelial cells were scraped and incubated with TrypLE Express Enzyme at 37 °C for 30 min to dissociate. Dissociated limbal epithelial cells were stained with fluorescence conjugated antibodies for 30 min on ice (2.5μg/ml mouse anti-ABCB5 monoclonal antibody (mAb) (clone 3C2-1D12) conjugated with Alexa Fluor 647, 0.125μg/ml PE-conjugated anti-CD45 mAb). The cells were incubated with 30nM SYTOX Green Dead Cell Stain to exclude nonviable cells. Cell sorting was performed using FACSAria II cell sorter (BD Biosciences, San Jose, CA) using a 100 mm nozzle. The cells were loaded onto a Chromium chip (10X Genomics) followed by encapsulation in lipid droplets.
cDNA libraries were prepared per manufacturer's protocol (10X Genomics). cDNA libraries were sequenced using an Illumina NextSeq 500.

tag: cell type

value: FACS sorted ABCB5-positive LSCs

value: LSCs


title: RNA_0h_KCl_rep1

description: total RNA


title: SBR22 Sham

tag: Sex


title: RNA-Seq_RPB11_IAA_rep2

value: mouse embryonic stem cells (V6.5)

value: RPB11 degron 12h mESCs


title: Y537N_1_042


title: SAB22 Sham

tag: age


title: Y537N_1_041


title: RNA-Seq_RPB11_IAA_rep1


title: SBE28 Sham


title: RNA-Seq_RPB11_Untreated_rep2

value: RPB11 untreated mESCs


title: ABCB5-positive LSCs from donor 2


title: ABCB5-positive LSCs from donor 1


title: Y537N_1_040


title: CAB29 Coarctation

value: Coarctation


title: SAB28 Sham

value: Spargue Dawley


title: RNA-Seq_RPB10_IAA_rep2

value: RPB10 degron 12h mESCs


title: CBR27 Coarctation

value: SD/Rij

value: Male


title: GB4649 KO3 TGFb+IL4

city: Bethesda

first: Keji

last: Zhao

state: MD

address: Building 10 Room 7B06A

department: National Heart, Lung, and Blood Institute

institute: national Institute of Health

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt

data_processing:
Basecalls performed
Reads from DNase-seq data were mapped to mouse genome (mm9) using Bowtie2. Reads with MAPQ ≤ 10 or redundant reads that mapped to the same location and orientation were removed. DNase-seq data was normalized by the total library size. DHS from DNase-seq data were called by MACS as ‘narrow peaks’. Reads from RNA-seq data were mapped to mouse genome (mm9) using Bowtie2. Gene expression level was then measured by calculating the normalized reads per kilobase per million mapped reads (RPKM) for each gene.
Genome_build: mm9
Supplementary_files_format_and_content: KZ1410_KZ1411_rpkm_table.txt: A table contains the rpkm values. First Column) Gene, other columns) RPKM values for the RNA-seq data
Supplementary_files_format_and_content: KZ1344_GB1665_macs_peaks.narrowPeak:Peak file from the output of the software macs2. First column) chromosome, Second column) Start, Third column)end, Fourth Column)name, Fifth column)score, Sixth column)strand, Seventh column) signal-value, Eighth column) p-value, ninth column) q-value tenth column) peak
Supplementary_files_format_and_content: KZ1344_GB1666_macs_peaks.narrowPeak:Peak file from the output of the software macs2. First column) chromosome, Second column) Start, Third column)end, Fourth Column)name, Fifth column)score, Sixth column)strand, Seventh column) signal-value, Eighth column) p-value, ninth column) q-value tenth column) peak
Supplementary_files_format_and_content: KZ1344_GB1667_macs_peaks.narrowPeak:Peak file from the output of the software macs2. First column) chromosome, Second column) Start, Third column)end, Fourth Column)name, Fifth column)score, Sixth column)strand, Seventh column) signal-value, Eighth column) p-value, ninth column) q-value tenth column) peak
Supplementary_files_format_and_content: KZ1344_GB1668_macs_peaks.narrowPeak:Peak file from the output of the software macs2. First column) chromosome, Second column) Start, Third column)end, Fourth Column)name, Fifth column)score, Sixth column)strand, Seventh column) signal-value, Eighth column) p-value, ninth column) q-value tenth column) peak

extract_protocol:
Cell were sorted directly into 700 ml of QIAzol Lysis Reagent in the miRNAeasy Micro Kit (QIAGEN, 217084). Total RNA was extracted and on-column digestion with DNase (QIAGEN, 79254) was performed. RNA-seq libraries were prepared through the Smart-seq2 method 50 as previously described (Hu, G. et al. Transformation of Accessible Chromatin and 3D Nucleome Underlies Lineage Commitment of Early T Cells. Immunity 48, 227-242 e228, doi:10.1016/j.immuni.2018.01.013 (2018)).
libraries prepared and sequenced as described for the DNase-seq assays

tag: cell lines

value: CD4 primary cell


title: RNA_0h_KCl_rep2

description: total RNA

tag: tag

value: hESC-derived neurons


title: RNA-Seq_RPB12_Untreated_rep2

value: RPB12 untreated mESCs


title: RNA-Seq_RPB11_Untreated_rep1


title: Y537N_1_039


title: GB4641 KO1 TGFb+IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: Y537C_2_132


title: CBE27 Coarctation

value: 10-15 weeks

value: Aquavive water (Innovive)

value: Chow diet


title: Y537N_1_037

value: MCF7-Luc cells


title: RNA-Seq_RPB10_IAA_rep1


title: CAB27 Coarctation

value: Envigo Teklad 22/5 #8640

value: 15 weeks


title: Y537N_1_038


title: SBR26 Sham

value: Sham


title: GB4648 KO3 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: GB4645 KO2 TGFb+IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: Y537C_2_130


title: SBE26 Sham


title: RNA-Seq_RPB10_Untreated_rep1

value: mouse embryonic stem cells (V6.5)

value: RPB10 untreated mESCs


title: SAB26 Sham


title: RNA-Seq_RPB9_IAA_rep1

value: RPB9 degron 12h mESCs


title: GB4647 KO3 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: CBE25 Coarctation

value: Coarctation

value: Spargue Dawley


title: Y537C_2_129


title: RNA-Seq_RPB9_Untreated_rep2

value: RPB9 untreated mESCs


title: GB4643 KO2 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB10_Untreated_rep2


title: Y537C_2_128


title: GB4639 KO1 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: CAB25 Coarctation

value: SD/Rij

value: Male


title: Y537C_2_127


title: SBR24 Sham


title: GB4644 KO2 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt

value: CD4 primary cell


title: RNA-Seq_RPB9_IAA_rep2


title: Y537C_1_126


title: RNA-Seq_RPB8_IAA_rep1

value: RPB8 degron 12h mESCs


title: RNA-Seq_RPB9_Untreated_rep1


title: SBE24 Sham

value: 10-15 weeks


title: RNA-Seq_RPB8_IAA_rep2


title: GB4640 KO1 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: GB4642 KO2 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: GB4646 KO3 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: Y537C_2_131


title: Y537C_1_125


title: SAB24 Sham

value: Aquavive water (Innovive)


title: Y537C_1_124


title: CBR23 Coarctation

value: Chow diet


title: GB4638 KO1 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB8_Untreated_rep2

value: RPB8 untreated mESCs


title: RNA-Seq_RPB8_Untreated_rep1


title: CBE23 Coarctation

value: Envigo Teklad 22/5 #8640


title: Y537C_1_123

value: MCF7-Luc cells


title: GB4637 WT3 TGFb+IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB7_IAA_rep2

value: RPB7 degron 12h mESCs


title: GB4633 WT2 TGFb+IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: Y537C_1_122


title: CAB23 Coarctation

value: 15 weeks


title: GB4629 WT1 TGFb+IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB7_IAA_rep1

value: mouse embryonic stem cells (V6.5)


title: GB4636 WT3 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: L536R_3_114


title: Y537C_1_121


title: RNA-Seq_RPB7_Untreated_rep2

value: RPB7 untreated mESCs


title: SBR20 Sham

value: Sham


title: SBE20 Sham

value: Spargue Dawley


title: L536R_3_113


title: RNA-Seq_RPB7_Untreated_rep1


title: GB4632 WT2 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: GB4628 WT1 IL4

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB6_IAA_rep2

value: RPB6 degron 12h mESCs


title: RNA-Seq_RPB6_IAA_rep1


title: GB4635 WT3 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: CBR19 Coarctation

value: Coarctation

value: SD/Rij


title: L536R_3_111


title: CBE19 Coarctation

value: Male


title: L536R_3_112


title: GB4631 WT2 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt

value: CD4 primary cell


title: RNA-Seq_RPB6_Untreated_rep2

value: RPB6 untreated mESCs


title: L536R_3_110


title: CAB19 Coarctation

value: 10-15 weeks

value: Aquavive water (Innovive)


title: RNA-Seq_RPB6_Untreated_rep1


title: GB4627 WT1 TGFb

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: L536R_3_109


title: RNA-Seq_RPB5_IAA_rep2

value: RPB5 degron 12h mESCs


title: GB4634 WT3 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: L536R_2_108


title: SAB20 Sham

value: Chow diet

value: Envigo Teklad 22/5 #8640


title: GB4630 WT2 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB5_IAA_rep1


title: RNA-Seq_RPB5_Untreated_rep2

value: RPB5 untreated mESCs


title: L536R_2_105

value: MCF7-Luc cells


title: GB1668  Dnase I-seq mouse TGF+IL4 KO3 Tamoxifen-treated

description:
Dnase-seq
KZ1344_GB1668_macs_peaks.narrowPeak

extract_protocol:
Cells were crosslinked with 1 % formadehyde at room temperature for 10 min. 0.3 unit of DNase I (Roche, 04716728001) was added to the cells and incubated at 37 C for 5 min. The reaction was stopped by adding 10 mM EDTA. The DNase-seq libraries were prepared by the method as previously described(Fu, Y. et al. STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus. Nat Commun 11, 4882, doi:10.1038/s41467-020-18648-6 (2020)).
Purified DNA was end-repaired using end-repair kit (ER81050, Epicentre), followed by A-addition using Klenow fragment (3’ to 5’ exo minus) and dATP. The Y-shaped adaptors were then ligated to DNA ends using T4 DNA ligase. Libraries were amplified with Illumina primers for 16 cycles and library fragments from 150bp to 300bp were isolated for sequencing on Illumina HiSeq or NovaSeq platforms.


title: RNA-Seq_RPB5_Untreated_rep1

value: mouse embryonic stem cells (V6.5)


title: L536R_2_104


title: RNA-Seq_RPB4_IAA_rep2

value: RPB4 degron 12h mESCs


title: GB1667  Dnase I-seq mouse TGF+IL4 WT3 Oil-treated

description:
Dnase-seq
KZ1344_GB1667_macs_peaks.narrowPeak


title: GB4626 WT1 Control

description:
RNA-seq
KZ1410_KZ1411_rpkm_table.txt


title: RNA-Seq_RPB4_IAA_rep1


title: GB1665 Dnase I-seq mouse Contorl WT3 Oil-treated

description:
Dnase-seq
KZ1344_GB1665_macs_peaks.narrowPeak


title: GB1666  Dnase I-seq mouse Control KO3 Tamoxifen-treated

description:
Dnase-seq
KZ1344_GB1666_macs_peaks.narrowPeak


title: RNA-Seq_RPB4_Untreated_rep2

value: RPB4 untreated mESCs


title: L536R_1_120


title: RNA-Seq_RPB4_Untreated_rep1


title: ChIP_ndx-gfp_ip_rep2

description:
A. thaliana reference genome (TAIR10)
Paired-end ChIP-seq; reads1 and reads2 in different raw files (replica 2); ip (fastq), IP/input (bigWig).
Antibody against GFP epitope was used anti-GFP antibody - #ab290 Abcam.

data_processing:
Sequenced reads were aligned to the A. thaliana reference genome (TAIR10; NCBI; Ecotype: Columbia-0) using the bowtie2 algorithm with the default parameters.
Samtools (REF) was used for making .bam files and indexing. Low mapping quality and PCR-duplicated reads were omitted from downstream analysis (http://broadinstitute.github.io/picard/). Deeptools2 bamCoverage was used to create RPKM (Reads Per Kilobase per Million mapped reads) normalized coverage files (.bedgraph and .bigwig). Read densities were calculated for 20 bp bins (--binSize 20 --operation ratio --smoothLength 60 –normalizeUsing RPKM) and IP/input bigwig ratios were made by bigwigCompare.
MACS2 was used with default settings to identify ChIP peaks in flag-NDX and NDX-GFP samples.
Assembly: A. thaliana reference genome (TAIR10; NCBI; Ecotype: Columbia-0).
Supplementary files format and content: bigwig files are from the IP/input ratio.
Supplementary files format and content: bed files contain the processed peaks.

growth_protocol: 2 g of 10-day-old flag-NDX (flag-NDX / ndx1-1 (FRI) / flc-2), NDX-GFP (NDX-GFP / ndx1-1(FRI) / flc-2), and ndx1-4 seedlings (no tag control) were harvested.

extract_protocol:
Plants were cross-linked under a vacuum chamber by phosphate buffered saline (PBS) containing 1% formaldehyde for 2x5 minutes. Excess formaldehyde was quenched by adding glycine to a final concentration of 0.125 M. Chromatin was purified and resuspended in Nuclei lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 20mM Hepes KOH pH 7.4, 10mM MgCl2, 0.5% Triton X-100, 5mM DTT and Protease Inhibitors) followed by Proteinase K treatment (Thermo Fisher Scientific) and crosslink-reversal in the same step, at 65°C, overnight. Total nucleic acid was prepared by phenol-chloroform extraction and precipitated with 2-volumes of ice cold isopropanol and 1/10 volume 4M ammonium-acetate. Samples were then divided into two equal parts: half of the samples were treated with 8 µl of RNaseH (5000 U/ml, New England Biolabs) at 37°C, overnight. Nucleic acid preps were diluted 4-fold in a ChIP dilution buffer (16.7 mM Tris-HCl pH 8, 1.2 mM EDTA, 1.1% Triton X-100, 167 mM NaCl) and DNA was fragmented by sonication to an average fragment size of 250-500 bp (low intensity, 3 x 5 min; 30 sec ON, 30 sec OFF, Bioruptor UCD-300, Diagenode). Dynabeads Protein A magnetic beads (ThermoFisher Scientific) were pre-blocked with PBS/EDTA containing 0.5% BSA. To immobilize the S9.6 antibody, 50 µl pre-blocked Dynabeads Protein A slurry was incubated with 10 μg of S9.6 antibody in IP buffer (50 mM Hepes/KOH at pH 7,5; 0,14 M NaCl; 5 mM EDTA; 1% Triton X-100; 0,1% Na-Deoxycholate, ddH2O) at 4°C for 4 hours, with rotation. Six micrograms of fragmented genomic DNA were added to the mixture and gently rotated at 4°C, overnight. Beads were washed two times with 1 ml of ChIP lysis buffer (low salt,50 mM Hepes/KOH pH 7.5, 0.14 M NaCl, 5 mM EDTA pH 8, 1% Triton X-100, 0.1% Na-Deoxycholate), 1 ml of high-salt ChIP lysis buffer (50 mM Hepes/KOH pH 7.5, 0.5 M NaCl, 5 mM EDTA pH 8, 1% Triton X-100, 0.1% Na-Deoxycholate), 1 ml of wash buffer (10 mM Tris-HCl at pH 8, 0.25 M LiCl, 0.5% NP-40, 0.5% w/v sodium deoxycholate, 1 mM EDTA at pH 8) and 1 ml of TE buffer (100 mM Tris-Cl at pH 8, 10 mM EDTA at pH 8) at 4°C. Elution was performed in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) for 15 min at 65°C. After purification by a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).
ChIP-seq libraries were prepared according to Illumina’s TruSeq ChIP Sample Preparation protocol except that the uracil tolerant KAPA HiFi HotStart DNA Polymerase (Kapa Bio) was used. Briefly, the enriched ChIP DNA samples were end-repaired and indexed adapters were ligated to the inserts. Purified ligation products were then amplified by PCR. Libraries were sequenced using 150 nt paired end read with Illumina HiSeq 2500 (EMBL Genomics Core Facility, Heidelberg, Germany).

treatment_protocol: For ChIP, antibodies against flag and GFP epitopes were used (anti-FLAG antibody - #2044 New England Biolabs; anti-GFP antibody - #ab290 Abcam).

value: Seedling

value: 10 days

value: NDX-GFP (genotype:NDX-GFP /ndx1-1(FRI) / flc-2)

tag: chip antibody

value: anti-GFP antibody


title: L536R_1_119


title: L536R_2_103


title: RNA-Seq_RPB3_IAA_rep2

value: RPB3 degron 12h mESCs


title: L536R_2_106


title: ChIP_ndx-gfp_ip_rep1

description:
A. thaliana reference genome (TAIR10)
Paired-end ChIP-seq; reads1 and reads2 in different raw files (replica 1); ip (fastq), IP/input (bigWig).
Antibody against GFP epitope was used anti-GFP antibody - #ab290 Abcam.


title: L536R_1_118


title: L536R_2_107


title: L536R_1_117


title: RNA-Seq_RPB3_Untreated_rep2

value: RPB3 untreated mESCs


title: ChIP_flag-ndx_ip_rep1

description:
A. thaliana reference genome (TAIR10)
Paired-end ChIP-seq; reads1 and reads2 in different raw files (replica 1); ip (fastq), IP/input (bigWig).
Antibody against flag epitope was used anti-FLAG antibody - #2044 New England Biolabs.

value: flag-NDX (genotype:flag-NDX / ndx1-1(FRI) / flc-2)

value: anti-FLAG antibody


title: L536R_1_116


title: ChIP_flag-ndx_input

description:
A. thaliana reference genome (TAIR10)
Paired-end ChIP-seq; reads1 and reads2 in different raw files; input (fastq).


title: RNA-Seq_RPB3_Untreated_rep1


title: RNA-Seq_RPB2_IAA_rep2

value: RPB2 degron 12h mESCs


title: MCF7_Luc_077

value: WT


title: L536R_1_115


title: RNA-Seq_RPB2_IAA_rep1


title: MCF7_Luc_078


title: MCF7_Luc_076


title: RNA-Seq_RPB2_Untreated_rep2

value: RPB2 untreated mESCs


title: RNA-Seq_RPB2_Untreated_rep1


title: ChIP_flag-ndx_ip_rep2

description:
A. thaliana reference genome (TAIR10)
Paired-end ChIP-seq; reads1 and reads2 in different raw files (replica 2); ip (fastq), IP/input (bigWig).
Antibody against flag epitope was used anti-FLAG antibody - #2044 New England Biolabs.


title: RNA-Seq_RPB3_IAA_rep1


title: RNA-Seq_RPB1_IAA_rep2

value: RPB1 degron 12h mESCs


title: MCF7_Luc_075


title: cells treated with neomycin sulfate 4 hours_biological rep 3

city: MADRID

first: Teresa

last: Fernandez-Acero

address: PLAZA DE RAMÓN Y CAJAL S/N

department: Departamento de Microbiología II

country: Spain

institute: Facultad de Farmacia UCM

description:
BY SN 3
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.

hyb_protocol: Following Affymetrix hybridization instructions.

scan_protocol: Following Affymetrix scanning instructions.

data_processing: CEL files from scanner were analyzed using GCRMA algorithm from Bioconductor software.

organism: Saccharomyces cerevisiae BY4741

source_name: yeast

label_protocol: Following Affymetrix labelling instructions.

growth_protocol: Saccharomyces cerevisiae strain BY4741 was grown overnight in YPD and diluted to 0.3 OD with fresh medium. Cultures were incubated for 4 hours at 30°C with or without neomycin sulfate.

extract_protocol: Total RNA was isolated from exponentially growing cells by the “mechanical disruption protocol” using the RNeasy kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. RNA concentrations were determined by measuring absorbance at 260 nm. RNA purity and integrity were assessed using RNA Nano Labchips in an Agilent 2100B Bioanalyzer (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.

treatment_protocol: Neomycin sulfate was added at 10 mg/ml and cells were incubated for 4 hours.

value: neomycin sulfate

tag: time point

value: 4 hours


title: RNA-Seq_RPB1_IAA_rep1

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

institute: Peking University

value: mouse embryonic stem cells (V6.5)


title: MCF7_Luc_074

value: MCF7-Luc cells

tag: mutation status (esr1)


title: MCF7_Luc_073


title: control cells 4 hours_biological rep 2

description:
BY 2
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.

value: control


title: control cells 4 hours_biological rep 1

description:
BY 1
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.


title: RNA-Seq_RPB1_Untreated_rep2

value: RPB1 untreated mESCs


title: MCF7_Luc_006


title: RNA-Seq_RPB1_Untreated_rep1


title: cells treated with neomycin sulfate 4 hours_biological rep 2

description:
BY SN 2
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.


title: cells treated with neomycin sulfate 4 hours_biological rep 1

description:
BY SN 1
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.


title: MCF7_Luc_005


title: RNA-Seq_WT_IAA_rep2

value: wide-type degron 12h mES


title: MCF7_Luc_004


title: K3

city: Newark

first: Tania

last: Wong

state: New Jersey

address: 205 South Orange Avenue

department: Microbiology, Biochemistry and Molecular Genetics

institute: Rutgers NJ Medical School

description: Bacterial (MKP103) RNA extracted from pure bacterial culture grown in vitro

data_processing:
basecalling using bcl2fastq  '2.20.0'
alignment using star aligner  '2.7.3a'
filtering using samtools (1.9), duplicates were marked using picard '2.22.3'; gatk = '4.2.0.0'
read quantification for mouse samples using featurecounts (M1,M2,M3,M5,M7,M8,M9,M10,M11,M14 and M16); read quantification for host samples using kallisto (K1, K2 and K3); subread = '1.6.3'
differential gene expression analysis using DEseq2 in Rstudio; R = '3.5.3',
Genome_build: mouse: GRCm38 fasta and gtf from Gencode vM21
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Klebsiella pneumoniae subsp. pneumoniae

source_name: MKP103 grown in Luria broth

growth_protocol: MKP103 was grown in Luria broth prior to bacterial RNA extraction

extract_protocol:
As per manuscript
According to the manufacturer’s instructions, Universal Prokaryotic RNA-Seq Prokaryotic AnyDeplete kit (NuGEN #0363-32)

treatment_protocol: Total RNA (host and bacteria) were extracted from murine lungs at 48 h post infection

value: bacterial culture

tag: infection status

tag: mouse genotype

tag: bacterial genotype


title: MCF7_Luc_003

tag: cell line


title: RNA-Seq_WT_Untreated_rep2

tag: treatment

value: wide-type untreated mES


title: K2

tag: tissue


title: control cells 4 hours_biological rep 3

description:
BY 3
All the procedures for microarrays experiments were carried out following the instructions of Affymetrix for each of the steps.


title: RNA-Seq_WT_Untreated_rep1


title: MCF7_Luc_001


title: MCF7_Luc_002


title: Hotfish

status: Public on Aug 08 2022

city: Seattle

first: Michael

last: Dorrity

state: Washington

address: 3720 15th Ave NE, Foege Building, Room S313

institute: University of Washington

description: single-cell RNA sequencing library containing hash molecules denoting where a nucleus originated from and the transcriptome measured for each cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were performed using the Brotman Baty sci-RNA-seq demultiplexing and processing pipelines (https://github.com/bbi-lab/bbi-dmux; https://github.com/bbi-lab/bbi-sci).
Count matrix was loaded into Monocle3 for further analysis
Assembly: GRCz11
Supplementary files format and content: hotfish.cds (monocle3 object)
Supplementary files format and content: HF4_counts.RDS (gene by cell counts, sparse matrix)
Supplementary files format and content: HF4_cell_metadata.txt (cell metadata, tab separated)
Supplementary files format and content: HF4_gene_metadata.txt (gene metadata, tab separated)
Supplementary files format and content: HF4_hash_sampleSheet.txt (hash sample table, tab separated)
Supplementary files format and content: HF4_processed_hashTable.csv (hash count table, tab separated)

source_name: Whole embryo

growth_protocol: Zebrafish embryos were raised in embryo media at 28.5, 32, and 34C.

extract_protocol:
Embryos were transferred, one by one, into separate wells of a 96-well V-bottom plate containing 75μL of 1X TrypLE + 2 mg/mL Collagenase P. Embryos were then dissociated by manual trituration at 30℃ once every 5 minutes. Dissociation continued until no visible chunks were present under a dissecting scope, which took between 20-40 minutes depending on embryo stage (e.g. 20 minutes for 18hpf and 40 minutes for 72hpf). Stop solution [1x dPBS, 5% FBS ] was then added to each well to quench the proteases.
3' end capture of polyAdenylated transcripts. In situ reverse transcription with barcoded RT primers followed by second strand synthesis, tagmentation and index PCR gives each nucleus and all the RNA molecules contained within it, a unique combination of barcode sequences. These barcodes are then demultiplexed to create single cells in silico.
snRNA-seq

value: Whole embryo

tag: developmental stage

tag: strain

value: AB

value: wild type

value: temperature perturbation experiment (controls, treated)

tag: temperature

tag: fraction

value: nuclear polyA RNA


title: Week 0 - replicate 1

city: Edmonton

first: Jason

middle: R.

last: Plemel

state: Alberta

address: 5-64 Heritage Medical Research Centre

department: Department of Medicine

institute: University of Alberta

data_processing:
The barcode processing and gene alignment was done according to the STAR method using the 10x cell ranger (v3.0.0) pipeline.
All the quality control was performed using Seurat v4.
Assembly: mm10
Supplementary files format and content: Comma-separated values files and matrix files

source_name: Corpus callosum

extract_protocol:
Mice were euthanized with Euthanyl Reg and transcardially perfused with ice-cold HBSS (Invitrogen 14175103). Brains were dissected, processed, and diced in ice-cold HBSS. Collected tissue was then dounced 5 times with a loose fitted dounce and subsequently filtered through 70 and 20μm cell strainers.  Cells were pelleted and myelin debris was removed with Debris Removal Solution (Miltenyi Biotech 130-109-398).  Cells were then blocked for 5 minutes with Anti-Mo CD16/CD32 (Invitrogen 14-0161-85) and stained using Zombie Aqua Fixable Viability Kit (Biolegend 423102), DRAQ5 Fluorescent Probe Solution (Thermofisher 62254) and FITC Mouse anti Rat CD11b (BD Biosciences 561684) for 20 min. After cell staining, cells were sorted on a Sony MA900.  Live nucleated single cells that were CD11b positive were gated and collected.  Final collected cell counts were determined using a hemocytometer.
The FACS-isolated cells were processed for single cell sequencing on the 10x Genomics  Chromium Controller (PN-1000121) using the 10x Genomics Chromium Next GEM Single Cell 3’ GEM, Library and Gel Bead Kit v3.1 (PN-1000121), following the manufacturer’s instructions and sequenced on the Illumina HiSeq P150 sequencer (Novogene). We chose the paired end, single indexing form of sequencing with an average read depth of 50,000 reads per cell.

tag: cell type

value: Microglia and Macrophages

value: Corpus callosum

tag: Sex

value: Male

value: 13 weeks

value: not treated


title: Week 5 - replicate 2

tag: age

value: Cuprizone


title: Week 0 - replicate 2


title: RNA-Seq_WT_IAA_rep1

city: Beijing

value: mouse embryonic stem cells (V6.5)


title: RMB765

city: Singapore

first: jinmiao

last: chen

address: 8A Biomedical Grove, #04-06 Immunos

country: Singapore

institute: Singapore Immunology Network

data_processing:
Raw reads were aligned to the mouse reference genome GRCm38 downloaded from GENCODE using RSEM program version 1.2.19 with default parameters.
Gene expression values in transcripts per million (TPM) were calculated using the RSEM program and the mouse GENCODE gene annotation version M4.
Quality control and outlier cell detection was performed using Seurat R package.
Assembly: GRCm38
Supplementary files format and content: tab delimited TPM table

extract_protocol:
single cells were captured on Fluidigm C1 mRNA Seq IFC and lysed and reverse-transcribed in situ
Nextera XT


title: RMB768


title: RMB769


title: RMB764


title: RMB770


title: RAW264.7, low sodium 3

city: Washington

first: Julianna

last: Barsony

state: DC

address: 4000 Reservoir Rd D/233

institute: Georgetown University

description: 4 biological replicates

hyb_protocol: Samples were hybridized to GeneChip Mouse Genome 430 2.0 arrays containing 45,101 probe sets corresponding to known genes and expressed sequence tags (Affymetrix)

scan_protocol: GeneScript 3000 Scanner enabled for High-Resolution Scanning and Gene Chip Operating Software

data_processing: Microarray data were normalized and analyzed using the Affymetrix Gene Chip software, Microarray Analysis Suite 5.0. Normalized expression values were compared between groups by anova using the Partek Pro software (Partek)

source_name: RAW264.7 osteoclastic cell line, low sodium

label_protocol: 5 µg RNA from each sample was converted into biotin-labeled cDNA using reagents and protocol from Affymetrix (Thermo Fisher Scientific)

growth_protocol: Low passage number murine monocytic RAW264.7 cells were obtained from the American Type Culture Collection, Manassas, VA. Cells were all grown in Minimal Essential Media Alpha with glucose 4.5 g/L (αMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (ATCC), 100µg/ml penicillin/streptomycin (Invitrogen) and 2mM glutamine (normal medium; [Na+] = 137 mmol/l)).

extract_protocol: Total RNA was extracted using PureLink kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, and purified using the Rneasy Mini Kit (Qiagen, Germantown, MD)).

treatment_protocol: Experiments included 4 biological replicates of RAW264.7 cells and were placed into normal and another 4 biological replicates for low [Na+] media with cytokines (10 ng/mL recombinant mouse M-SCF and 30 ng/mL recombinant mouse receptor activator of NF-κB ligand (RANKL) (both from R&D Systems, Inc., Minneapolis, MN). Treatment was for 24 hours. To generate media with low [Na+] = 120 mmol/l concentration, the normal [Na+] medium was diluted with a mixture of water, minerals (except sodium), amino acid concentrate and vitamins (Mediatech Inc., Herndon, VA) to match the normal medium, while maintaining normal osmolality = 290 mOsm/L via the addition of mannitol.  Sodium and osmolality of media was measured before application to cells using a sodium analyzer (Coulter ELISE, Beckman, Fullerton, CA) and an osmometer (Model 3900, Advanced Instruments, Inc., Norwood, MA).

value: RAW264.7 osteoclastic cell line

value: low sodium


title: RMB763


title: RAW264.7, normal sodium 3

description: 4 biological replicates

source_name: RAW264.7 osteoclastic cell line, normal sodium

value: normal sodium


title: RAW264.7, low sodium 2

description: 4 biological replicates


title: K1

value: bacterial culture


title: RAW264.7, low sodium 1

description: 4 biological replicates


title: RMB762


title: RAW264.7, normal sodium 1

description: 4 biological replicates


title: RAW264.7, normal sodium 2

description: 4 biological replicates


title: Week 5 - replicate 1


title: RMB760


title: RMB759


title: RMB758


title: RMB756


title: RMB755


title: RMB757


title: RMB754


title: RMB752


title: RMB767


title: RMB753


title: RMB751


title: RMB766


title: RMB750


title: RMB749


title: RMB748


title: RMB747


title: RMB761


title: RMB746


title: RMB744


title: RMB741


title: RMB742


title: RMB739


title: RMB743


title: RMB738


title: RMB740


title: RMB734


title: RMB735


title: RMB736


title: RMB732


title: RMB727


title: RMB737


title: RMB728


title: RMB733


title: RMB726


title: RMB731


title: RMB724


title: RMB729


title: RMB723


title: RMB745


title: RMB720


title: RMB722


title: RMB725


title: RMB719


title: RMB718


title: RMB717


title: RMB715


title: RMB716


title: RMB713


title: RMB714


title: RMB711


title: RMB712


title: RMB730


title: RMB710


title: RMB708


title: RMB706


title: RMB709


title: RMB707


title: RMB705


title: RMB701


title: RMB699


title: RMB698


title: RMB721


title: RMB702


title: RMB704


title: RMB700


title: RMB697


title: RMB696


title: RMB694


title: RMB695


title: RMB693


title: RMB692


title: RMB690


title: RMB689


title: RMB691


title: RMB688


title: RMB606


title: RMB602


title: RMB604


title: RMB703


title: RMB601


title: RMB599


title: RMB603


title: RMB600


title: RMB598


title: RMB597


title: RMB596


title: RMB595


title: RMB594


title: RMB593


title: RMB592


title: RMB591


title: RMB590


title: RMB585


title: RMB605


title: RMB588


title: RMB586


title: RMB687


title: RMB583


title: RMB581


title: RMB580


title: RMB578


title: RMB587


title: RMB577


title: RMB575


title: RMB576


title: RMB573


title: RMB574


title: RMB572


title: RMB569


title: RMB570


title: RMB579


title: RMB566


title: RMB565


title: RMB589


title: RMB582


title: RMB584


title: RMB568


title: RMB562


title: RMB561


title: RMB560


title: RMB563


title: RMB558


title: RMB559


title: RMB557


title: RMB554


title: RMB555


title: RMB571


title: RMB551


title: RMB567


title: RMB553


title: RMB552


title: RMB549


title: RMB546


title: RMB545


title: RMB543


title: RMB548


title: RMB541


title: RMB542


title: RMB1483


title: RMB1484


title: RMB1482


title: RMB1479


title: RMB1480


title: RMB1478


title: RMB1477


title: RMB556


title: RMB1476


title: RMB550


title: RMB1474


title: RMB547


title: RMB1473


title: RMB1471


title: RMB1475