title: FT5, TCR

description: scTCR-seq

data_processing:
Data processing of single-cell RNA sequencing libraries: Cell Ranger v3.0.2 was used to demultiplex and map sequencing reads against the GRCh38 genome. The resulting count matrices were loaded into R using read10x function form Seurat R package. All downstream analyses were implemented using R v4.0.3 and the package Seurat v3.2.3 (Stuart et al., 2019). Low-quality and doublet cells were filtered. Percentage of mitochondrial genes were plotted as well, and outliers were removed to filter out dead cells. Cell cycle score was calculated for all cells and regressed out. Integration vignette from Seurat v3.0 was followed to generate merged Seurat objects using the SCTransform function and regressing mitochondrial genes and trdv & trgv genes. PCA component was performed using pca Seurat function with 20 dimensions. RunUMAP function was used to obtain bidimensional coordinates for each cell. We determined the k-nearest neighbors of each cell using the FindNeighbors function and used this knn graph to construct the SNN graph by calculating the neighborhood overlap (Jaccard index) between every cell and its k.param nearest neighbors. Finally, we used the FindClusters function to cluster cells using the Louvain algorithm based on the same PCs as RunUMAP function.
Single-cell TCR sequencing analysis: Sc TCR libraries were generated by using CellRanger vdj pipeline (v3.0.2). Human genome GRCh38 was used as reference for alignment. Integrated FT and PNT Seurat objects (gene expression data) were combined with their respective sc TCR-seq (TCR sequence) data based on shared 10× cell barcodes and following the script provided here (https://www.biostars.org/p/384640/). Only those cells expressing productive TCR sequences (γ and/or δ chain) from the ‘filtered_contig_annotations’ file were retained for data integration in the Seurat objects.
RNA-Seq samples: Approximately 30 million paired-end reads (2x101 bases) /sample were collected on a NovaSeq 6000 (Illumina). Paired-end reads with variable trimming (minimum size 36 bases) were mapped against the human reference genome (GRCh38) using STAR_2.5.3a software to generate read alignments for each sample. Annotations Homo_sapiens.GRCh38.90.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTSeq, followed by a library size normalization data processing step. Supplementary_files_format_and_content: CSV file include CPM (count per million) values for each Sample.
Bulk TCR-seq samples: Raw sequencing reads from fastq files (read 1 and read 2) were aligned to reference V, D and J genes from GenBank database specifically for ‘TRG’ or ‘TRD’ to build CDR3 sequences using the MiXCR software version 2.1.1. Default parameters were used except to assemble TRDD gene segment where 3 instead of 5 consecutive nucleotides were applied as assemble parameter. CDR3 sequences were then exported and analyzed using VDJtools software version 1.2.1 using default settings. Sequences out of frame and containing stop codons were excluded from the analysis. Finally data from both files, sorted Vg9Vd2 and nonVg9Vd2, were combined in a same csv file to analysis publicity levels in the single cell dataset.
Assembly: GRCh38
Supplementary files format and content: scRNA-seq: matrix.mtx, features.tsv, barcodes.tsv. // matrix files include transcript count from each single-cell GEX library, barcodes tsv files includes the barcodes present in that single-cell GEX library and features tsv file includes the ID of the genes detected in that single-cell GEX library. /-/ scTCR-seq: TCR_filtered_contig_annotations.csv, TCR_all_contig_annotations.csv. // CSV files include single-cell barcoded CDR3 sequences. /-/ bulk RNA-seq file: CPM_FT_PNT_Vg9Vd2.csv // CSV file include CPM (count per million) transcript values for each sample. /-/ bulk TCR-seq files: FT17_d.csv,FT17_g.csv,FT19_d.csv,FT19_g.csv,FT3_d.csv,FT3_g.csv,PT3_d.csv,PT3_g.csv,PT4_d.csv,PT4_g.csv,PT5_d.csv,PT5_g.csv,PT6_d.csv,PT6_g.csv,PT7_d.csv,PT7_g.csv,PT8_d.csvPT8_g.csv // Each csv file contains CDR3 sequences (gamma for files with a g in the name and delta for files with a d in the name) of sorted gamma delta T cells.
Library strategy: scTCR-seq

extract_protocol:
Cell suspensions from fetal thymus and pediatric thymus samples were obtained as previously described (Tieppo et al. JEM 2020) and cryopreserved in liquid nitrogen with freezing media (10%DMSO, 90%FBS). When samples were used, cells were thawed and washed twice with RPMI 1640 (Gibco) complete medium, labeled with Zombie NIR dye (BioLegend), and then subsequently stained for cell surface Ags with Abs directed against CD3 (clone UCHT1; BD Biosciences), TCR-γδ (11F2; BD Biosciences), TCR-Vγ9 (IMMU360; Beckman Coulter), TCR-Vδ2 (IMMU389; Beckman Coulter). For the sc experiment, CD3+ γδTCR+ were sorted for “γδ” thymocytes (mean purity 98% of living cells) on a FACS Aria III (BD Biosciences). Libraries for sc RNA and TCR sequencing were generated from 1.5-2*104 FACS-sorted γδ thymocytes using the Chromium Single Cell 5’ Library Gel Bead and Construction kit as well as Chromium Single Cell V(D)J Enrichment Kit (10x Genomics, CA, USA) according to the user guidelines (v1 Chemistry, Single Cell V(D)J protocol number CG000086). Single-cell TCR and gene expression libraries were generated according to Chromium Single Cell V(D)J protocol (10x Genomics). 2μL of cDNA amplified and purified from GEMs (“Gel bead in EMulsion” droplets) were used to amplify γδTCR CDR3 sequences. Custom primers specific for TRDC and TRGC constant gene segments were designed for this purpose. The remaining of cDNA was used for gene expression library construction according to 10X Genomics protocol instructions. Agilent Bioanalyzer High Sensitivity DNA chips were used to check quality control read-outs of scRNA-seq and scTCR-seq libraries using a Bioanalyzer 2100 machine (Agilent Technologies). The scRNA-seq libraries were sequenced on the Illumina NovaSeq 6000 platfotm from BRIGHTcore (Brussels University Alliance).
Cell suspensions from fetal thymus and pediatric thymus samples were obtained as previously described (Tieppo et al. JEM 2020) and cryopreserved in liquid nitrogen with freezing media (10%DMSO, 90%FBS). When samples were used, cells were thawed and washed twice with RPMI 1640 (Gibco) complete medium, labeled with Zombie NIR dye (BioLegend), and then subsequently stained for cell surface Ags with Abs directed against CD3 (clone UCHT1; BD Biosciences), TCR-γδ (11F2; BD Biosciences), TCR-Vγ9 (IMMU360; Beckman Coulter), TCR-Vδ2 (IMMU389; Beckman Coulter). For the sc experiment, CD3+ γδTCR+ were sorted for “γδ” thymocytes (mean purity 98% of living cells) on a FACS Aria III (BD Biosciences). Libraries for sc RNA and TCR sequencing were generated from 1.5-2*104 FACS-sorted γδ thymocytes using the Chromium Single Cell 5’ Library Gel Bead and Construction kit as well as Chromium Single Cell V(D)J Enrichment Kit (10x Genomics, CA, USA) according to the user guidelines (v1 Chemistry, Single Cell V(D)J protocol number CG000086). Single-cell TCR and gene expression libraries were generated according to Chromium Single Cell V(D)J protocol (10x Genomics). 2μL of cDNA amplified and purified from GEMs (“Gel bead in EMulsion” droplets) were used to amplify γδTCR CDR3 sequences. Custom primers specific for TRDC and TRGC constant gene segments were designed for this purpose. The remaining of cDNA was used for gene expression library construction according to 10X Genomics protocol instructions. Agilent Bioanalyzer High Sensitivity DNA chips were used to check quality control read-outs of scRNA-seq and scTCR-seq libraries using a Bioanalyzer 2100 machine (Agilent Technologies). The scRNA-seq libraries were sequenced on the Illumina NovaSeq 6000 platfotm from BRIGHTcore (Brussels University Alliance).

value: 17 wk and 5 days gestation time

tag: group

value: barcoded CDR3 RNA sequences


title: FT3, TCR

status: Public on Aug 19 2022

type: SRA

city: Brussels

first: David

last: Vermijlen

state: non-US Resident

address: Boulevard de Triomphe, accès 2 (CP205/3)

department: Pharmacotherapy and Pharmaceutics

country: Belgium

institute: Université Libre de Bruxelles

description: scTCR-seq

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

source_name: ϒδ thymocytes

tag: cell type

value: Gamma delta T cell

tag: age

value: 15 wk and 2 days gestation time

tag: tissue

value: fetal thymus


title: FT6, TCR

description: scTCR-seq

value: 22 wk and 6 days gestation time


title: PNT3, TCR

description: scTCR-seq

value: 11 years

value: pediatric thymus


title: FT4, TCR

description: scTCR-seq

value: 16 wk and 2 days gestation time


title: PNT3, GEX

description: scRNA-seq

library_source: transcriptomic single cell

data_processing:
Data processing of single-cell RNA sequencing libraries: Cell Ranger v3.0.2 was used to demultiplex and map sequencing reads against the GRCh38 genome. The resulting count matrices were loaded into R using read10x function form Seurat R package. All downstream analyses were implemented using R v4.0.3 and the package Seurat v3.2.3 (Stuart et al., 2019). Low-quality and doublet cells were filtered. Percentage of mitochondrial genes were plotted as well, and outliers were removed to filter out dead cells. Cell cycle score was calculated for all cells and regressed out. Integration vignette from Seurat v3.0 was followed to generate merged Seurat objects using the SCTransform function and regressing mitochondrial genes and trdv & trgv genes. PCA component was performed using pca Seurat function with 20 dimensions. RunUMAP function was used to obtain bidimensional coordinates for each cell. We determined the k-nearest neighbors of each cell using the FindNeighbors function and used this knn graph to construct the SNN graph by calculating the neighborhood overlap (Jaccard index) between every cell and its k.param nearest neighbors. Finally, we used the FindClusters function to cluster cells using the Louvain algorithm based on the same PCs as RunUMAP function.
Single-cell TCR sequencing analysis: Sc TCR libraries were generated by using CellRanger vdj pipeline (v3.0.2). Human genome GRCh38 was used as reference for alignment. Integrated FT and PNT Seurat objects (gene expression data) were combined with their respective sc TCR-seq (TCR sequence) data based on shared 10× cell barcodes and following the script provided here (https://www.biostars.org/p/384640/). Only those cells expressing productive TCR sequences (γ and/or δ chain) from the ‘filtered_contig_annotations’ file were retained for data integration in the Seurat objects.
RNA-Seq samples: Approximately 30 million paired-end reads (2x101 bases) /sample were collected on a NovaSeq 6000 (Illumina). Paired-end reads with variable trimming (minimum size 36 bases) were mapped against the human reference genome (GRCh38) using STAR_2.5.3a software to generate read alignments for each sample. Annotations Homo_sapiens.GRCh38.90.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTSeq, followed by a library size normalization data processing step. Supplementary_files_format_and_content: CSV file include CPM (count per million) values for each Sample.
Bulk TCR-seq samples: Raw sequencing reads from fastq files (read 1 and read 2) were aligned to reference V, D and J genes from GenBank database specifically for ‘TRG’ or ‘TRD’ to build CDR3 sequences using the MiXCR software version 2.1.1. Default parameters were used except to assemble TRDD gene segment where 3 instead of 5 consecutive nucleotides were applied as assemble parameter. CDR3 sequences were then exported and analyzed using VDJtools software version 1.2.1 using default settings. Sequences out of frame and containing stop codons were excluded from the analysis. Finally data from both files, sorted Vg9Vd2 and nonVg9Vd2, were combined in a same csv file to analysis publicity levels in the single cell dataset.
Assembly: GRCh38
Supplementary files format and content: scRNA-seq: matrix.mtx, features.tsv, barcodes.tsv. // matrix files include transcript count from each single-cell GEX library, barcodes tsv files includes the barcodes present in that single-cell GEX library and features tsv file includes the ID of the genes detected in that single-cell GEX library. /-/ scTCR-seq: TCR_filtered_contig_annotations.csv, TCR_all_contig_annotations.csv. // CSV files include single-cell barcoded CDR3 sequences. /-/ bulk RNA-seq file: CPM_FT_PNT_Vg9Vd2.csv // CSV file include CPM (count per million) transcript values for each sample. /-/ bulk TCR-seq files: FT17_d.csv,FT17_g.csv,FT19_d.csv,FT19_g.csv,FT3_d.csv,FT3_g.csv,PT3_d.csv,PT3_g.csv,PT4_d.csv,PT4_g.csv,PT5_d.csv,PT5_g.csv,PT6_d.csv,PT6_g.csv,PT7_d.csv,PT7_g.csv,PT8_d.csvPT8_g.csv // Each csv file contains CDR3 sequences (gamma for files with a g in the name and delta for files with a d in the name) of sorted gamma delta T cells.

value: barcoded RNA


title: IL-4 reporter GFPpos medLN and MLN of two mice (hashtag labelled) 1

city: Erlangen

last: Voehringer

address: Wasserturmstrasse 3-5

department: Department of Infection Biology

country: Germany

institute: University Clinic Erlangen

description:
Cells of medLN and MLN were hashtag labelled and multiplexed
medLN_MLN_barcodes.tsv.gz
medLN_MLN_features.tsv.gz
medLN_MLN_matrix.mtx.gz

data_processing:
Alignment to mm10 and aggregation of GFPpos ("-1" added to barcode) and GFPneg samples ("-2" added to barcode) with cellranger v6.1.2
Assembly: mm10 (GRCm38)
Supplementary files format and content: Output Cellranger 5' Gene Expression: tsv, mtx. Contains only detected cellular barcodes. For Targeted Gene Expression samples, non-targeted genes are removed from the filtered matrix.

organism: Mus musculus

source_name: medLN and MLN

extract_protocol:
Single cell suspension to be prepared with10x Chromium Single Cell 5′ Solution (Gene Expression Ananlysis and TCR-specific VDJ library or hashtag library)
Cells were subjected to 10x Chromium Single Cell 5′ Solution v2 library prep kit for gene expression analysis and TCR-specific VDJ analysis or hashtag specific processing according to the manufacturer’s instructions

treatment_protocol: At day 10 after Nb infection lungs and LNs (medLN and MLN) of IL-4eGFP reporter (4get) mice were harvested. Lungs were perfused with PBS, cut into small pieces and digested with the commercial “Lung Dissociation kit” (Miltenyi) according to manufacturer’s instructions. Digested lungs and complete LNs were gently mashed through a 100 μm cell strainer. For lung cells a 40% percoll purification was applied and erythrocytes were lysed with ACK-buffer (0.15 M NH4Cl, 1 mM KHO3, 0.1 mM Na2EDTA). Then samples of all organs were treated with Fc-receptor blocking antibody (anti-CD16/32, clone 2.4G2, BioXCell, West Lebanon, NH) and stained with anti-CD4-Percp-Cy5.5 antibody (clone: RM4-5). IL-4eGFP+CD4+ cells or IL-4eGFP-CD4+ were sorted.

tag: strain

value: medLN and MLN

value: CD4+ T cells


title: FT5, GEX

description: scRNA-seq


title: Y16_ChIPseq_IgG

status: Public on Aug 18 2022

city: Houston

first: Yejing

last: Ge

state: Texas

department: Cancer Biology

country: USA

institute: The University of Texas, MD Anderson Cancer Center

description: Sample 16

library_source: genomic

data_processing:
For Sample 12-15, raw sequencing 150-bp paired-end reads were aligned to the mus musculus reference genome (version mm10 from Genome Reference Consortium GRCm38) using STAR 2.6.0b aligner. Aligned reads were quantified against the reference annotation (GRCm38) to obtain RPKM (transcripts per million reads) and raw counts using  HTSeq (python/3.6/anaconda3/bin/htseq-count, with options -m union -a 10 -s  no), respectively.
For Sample 16, libraries were sequenced using PE150 on a NovaSeq lane with about 25-30M reads output and trimmed to obtain PE150 reads and demultiplexed. Raw sequencing reads were aligned to themus musculus reference genome (version mm10 from Genome Reference Consortium GRCm38) using the Bowtie (https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.0.1/) pipeline with ( -n 1 -m 1 --best --strata ).
Assembly: mm10
Supplementary files format and content: Sample 12-15: tab-separated values (TSV) file, gene counts

molecule: genomic DNA

source_name: skin

growth_protocol: skin epithelium

extract_protocol:
For Sample 12-15, total RNA was extracted using Direct-zol RNA Miniprep Kit (Zymo, R2051)
For Sample 12-15, Use NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and NEBNext Ultra II Directional RNA Library Prep Kit NEB #E7760) for library preparation. For Sample 16, after tagmentation reactions with TnD transposase, libraries that passed QC were then pooled and sequenced.

treatment_protocol: For Sample 12-15, skin was removed and digested with dispase. The epidermis was peeled from dermis and grinded into powder in liquid nitrogen for RNA isolation; For Sample 16, skin was removed and digested with trpsin,purified based on sort markers indicated.

tag: cell types

value: skin epithelia

tag: genotype

tag: sort markers

value: DAPI-, CD45- and Integrin A6+


title: FT4, GEX

description: scRNA-seq


title: KL8_RNAseq_HFD_KO_rep1

description: Sample 14


title: IL-4 reporter GFPneg medLN and MLN of two mice (hashtag labelled) CellSurface hash 2

description:
Cells of medLN and MLN were hashtag labelled and multiplexed; #1 Biolegend ref 155861 mouse 1 med LN  Barcode Sequence ACCCACCAGTAAGAC   #2 Biolegend ref 155863 mouse 2 med LN Barcode Sequence GGTCGAGAGCATTCA  #3 Biolegend ref 155865 mouse 1 MLN  Barcode Sequence CTTGCCGCATGTCAT  #4 Biolegend ref 155867 mouse 2 MLN  Barcode Sequence AAAGCATTCTTCACG
medLN_MLN_barcodes.tsv.gz
medLN_MLN_features.tsv.gz
medLN_MLN_matrix.mtx.gz


title: FT3, GEX

description: scRNA-seq


title: FT6, GEX

description: scRNA-seq


title: IL-4 reporter GFPpos medLN and MLN of two mice (hashtag labelled) CellSurface hash 1


title: exvivo_FT_S1_vg9vd2_D, TCR

description:
bulk TCR-seq
FT19_d.csv

data_processing:
Data processing of single-cell RNA sequencing libraries: Cell Ranger v3.0.2 was used to demultiplex and map sequencing reads against the GRCh38 genome. The resulting count matrices were loaded into R using read10x function form Seurat R package. All downstream analyses were implemented using R v4.0.3 and the package Seurat v3.2.3 (Stuart et al., 2019). Low-quality and doublet cells were filtered. Percentage of mitochondrial genes were plotted as well, and outliers were removed to filter out dead cells. Cell cycle score was calculated for all cells and regressed out. Integration vignette from Seurat v3.0 was followed to generate merged Seurat objects using the SCTransform function and regressing mitochondrial genes and trdv & trgv genes. PCA component was performed using pca Seurat function with 20 dimensions. RunUMAP function was used to obtain bidimensional coordinates for each cell. We determined the k-nearest neighbors of each cell using the FindNeighbors function and used this knn graph to construct the SNN graph by calculating the neighborhood overlap (Jaccard index) between every cell and its k.param nearest neighbors. Finally, we used the FindClusters function to cluster cells using the Louvain algorithm based on the same PCs as RunUMAP function.
Single-cell TCR sequencing analysis: Sc TCR libraries were generated by using CellRanger vdj pipeline (v3.0.2). Human genome GRCh38 was used as reference for alignment. Integrated FT and PNT Seurat objects (gene expression data) were combined with their respective sc TCR-seq (TCR sequence) data based on shared 10× cell barcodes and following the script provided here (https://www.biostars.org/p/384640/). Only those cells expressing productive TCR sequences (γ and/or δ chain) from the ‘filtered_contig_annotations’ file were retained for data integration in the Seurat objects.
RNA-Seq samples: Approximately 30 million paired-end reads (2x101 bases) /sample were collected on a NovaSeq 6000 (Illumina). Paired-end reads with variable trimming (minimum size 36 bases) were mapped against the human reference genome (GRCh38) using STAR_2.5.3a software to generate read alignments for each sample. Annotations Homo_sapiens.GRCh38.90.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTSeq, followed by a library size normalization data processing step. Supplementary_files_format_and_content: CSV file include CPM (count per million) values for each Sample.
Bulk TCR-seq samples: Raw sequencing reads from fastq files (read 1 and read 2) were aligned to reference V, D and J genes from GenBank database specifically for ‘TRG’ or ‘TRD’ to build CDR3 sequences using the MiXCR software version 2.1.1. Default parameters were used except to assemble TRDD gene segment where 3 instead of 5 consecutive nucleotides were applied as assemble parameter. CDR3 sequences were then exported and analyzed using VDJtools software version 1.2.1 using default settings. Sequences out of frame and containing stop codons were excluded from the analysis. Finally data from both files, sorted Vg9Vd2 and nonVg9Vd2, were combined in a same csv file to analysis publicity levels in the single cell dataset.
Assembly: GRCh38
Supplementary files format and content: scRNA-seq: matrix.mtx, features.tsv, barcodes.tsv. // matrix files include transcript count from each single-cell GEX library, barcodes tsv files includes the barcodes present in that single-cell GEX library and features tsv file includes the ID of the genes detected in that single-cell GEX library. /-/ scTCR-seq: TCR_filtered_contig_annotations.csv, TCR_all_contig_annotations.csv. // CSV files include single-cell barcoded CDR3 sequences. /-/ bulk RNA-seq file: CPM_FT_PNT_Vg9Vd2.csv // CSV file include CPM (count per million) transcript values for each sample. /-/ bulk TCR-seq files: FT17_d.csv,FT17_g.csv,FT19_d.csv,FT19_g.csv,FT3_d.csv,FT3_g.csv,PT3_d.csv,PT3_g.csv,PT4_d.csv,PT4_g.csv,PT5_d.csv,PT5_g.csv,PT6_d.csv,PT6_g.csv,PT7_d.csv,PT7_g.csv,PT8_d.csvPT8_g.csv // Each csv file contains CDR3 sequences (gamma for files with a g in the name and delta for files with a d in the name) of sorted gamma delta T cells.
Library strategy: TCR-seq

extract_protocol:
Cell suspension from FT and PNT samples was obtained as previously described (McGovern et al., 2017; Van Coppernolle et al., 2009). γδ T cells from postnatal suspensions were isolated by positive selection using anti-TCRγδ magnetic activated cell sorting beads (Miltenyi Biotec) according to the manufacturer’s instruction. Thus, the γδ+ fraction was further sorted into CD3+TCRγδ+Vγ9+Vδ2+ subset (all around 10,000 cells) on a FACS Aria III cell sorter (BD Biosciences), snap-frozen in liquid nitrogen, and stored at −80°C for later RNA extraction. The mean purity was 95.0% (percentage of living cells). .RNA was extracted using RNeasy Plus Micro kit according to manufacturer’s instructions.
Prior steps before TCRγ (TRG) and TCRδ (TRD) high-throughput sequencing were performed as previously described (Tieppo et al. JEM 2020 and Papadopoulou et al. JI 2019). High-throughput sequencing of the generated amplicon products containing the TRG and TRD sequences was performed on an Illumina MiSeq platform using the V2 300 kit
RNA-Seq (RACE)

value: 19 wk gestation time


title: IL-4 reporter GFPneg medLN and MLN of two mice (hashtag labelled) 2


title: KL9_RNAseq_HFD_KO_rep2

description: Sample 15


title: NPC_RNAseq_ZIKVpos_24h

city: La Jolla

first: Aaron

middle: F

last: Carlin

state: California

address: 9500 Gilman Drive GPL Rm 118

department: Pathology and Medicine

institute: University of California, San Diego

description: Ribosome-depleted RNA-seq

data_processing:
Basecalling, demultiplexing, and adapter trimming was performed using Illumina's bcl2fastq (v2.17.1.14)
RNA-seq and csRNA-seq reads aligned to the GRCh38/hg38 assembly using STAR were used to generate gene expression FPKM values using HOMER. Gene expression (RNA-seq) was measured using HOMER with the following parameters: analyzeRepeats.pl rna hg38 -count exons -raw -pc 3 -strand - -condenseGenes followed by DESeq normalized regularized logarithm (rlog) normalization values while accounting for individual donors in the design matrix. Gene FPKM values were defined for primary Gencode transcripts using HOMER (v4.10) using only strand-specific (RNA-seq = exonic reads) reads for each transcript. csRNA-seq reads aligned to the GRCh38/hg38 assembly from three replicates per condition (mock, ZIKV-, ZIKV+) were combined and TSRs identified using HOMER findcsRNATSS.pl with the corresponding combined input and RNA-seq tag directories.
ChIP-seq reads were aligned to the human genome (hg38) using bowtie2 (Langmead and Salzberg, 2012), keeping only reads that map to a single unique genomic location for further analysis (MAPQ > 10). ChIP-seq peaks were called using tags from three replicates per condition with input DNA as background using HOMER’s getDifferentialPeaksReplicates.pl using the “-style factor” (5-fold enrichment over background and FDR < 0.001).
Assembly: GRCh38/hg38
Supplementary files format and content: RNA-seq tab-delimited text files include FPKM values for each sample. Tab-delimited BED files containing peak positions for each ChIP-seq experiment or combined replicates for csRNA-seq experiments.

source_name: human induced pluripotent cell line WT-126

growth_protocol: Human peripheral blood was separated using Histopaque 1077 and centrifuged at 300xg for 30 min at 4˚C. The buffy coat was washed once, and red blood cells were lysed with molecular grade water. Monocytes were negatively selected from the buffy coat using the human pan monocyte isolation kit (Miltenyi Biotec) as described by the manufacturer. Cells were seeded at 1.5 x 106 cells/mL onto polystyrene plates and differentiated for 7 days in hDC media composed of RPMI 1640 Medium, GlutaMAX™ Supplement, 1% penicillin/streptomycin, 2.5% HEPES buffer, and freshly added cytokines including 100 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and 100 ng/ml recombinant human interleukin 4 (rhIL-4). Cells were incubated at 37˚C, 5% CO2 and media was changed every 2-3 days.

extract_protocol:
For RNA-seq and csRNA-seq, after ZIKV infection of hDCs for 24 hours, cells were collected and stained with Zombie Violet™ Fixable Viability stain (BioLegend) for 20 min at 4˚C. After washing, cells were fixed and permeabilized with 4% paraformadehyde (PFA), 0.1% saponin in the presence of RNasin Ribonuclease inhibitor (400 U/mL). After centrifugation, samples were resuspended in wash buffer (0.2% BSA, 0.1% saponin and 400 U/mL of RNasin Ribonuclease inhibitor). Sample were blocked with human Fc blocker diluted in staining buffer (1% Bovine serum albumin (BSA), 0.1% saponin, 1600 U/mL RNasin Ribonuclease inhibitor) for 5 min, 4˚C. Then, 4G2-AF647 antibody was added to samples and incubated for 30 min, 4˚C. Samples were centrifuged at 1000xg for 3 min, 4˚C. Cells were washed using wash buffer and centrifuged at 1000xg for 3 min, 4˚C. Samples were resuspended in sort buffer containing 0.5% BSA and 1600 U/mL of RNase inhibitor in 1XPBS and sorted into ZIKV+ or DENV+ and ZIKV− or DENV- cells on a FACSAria cell sorter (BD Biosciences). After sorting, cells were pelleted at 4oC, supernatant discarded and total RNA isolated using Ambion’s RecoverAll Total Nucleic Acid Isolation Kit [AM1975], starting at the protease digestion step. All steps were performed per manufacturer recommendations with the following modification. Cells were incubated in digestion buffer for 3 hours at 50oC supplemented with RNasin Plus RNase Inhibitor. RNA was treated with in column DNase per manufacturer instructions, eluted and RNA quality determined by BioAnalyzer using the Eukaryote Total RNA Pico Chip. Samples with RIN values greater than 8.0 were used for library preparation. For chromatin immunoprecipitation-sequencing (ChIP-seq) experiments, moDCs were cross-linked with 4 mM disuccinimidyl glutarate (DSG) in PBS for 30 minutes at RT followed by 1% formaldehyde for 15 minutes at RT and quenched with 0.125 M glycine. Preparation of moDCs for FACS was performed as described above for RNA-seq except that 1x cOmplete protease inhibitors (Roche) were included in all buffers instead of RNasin Plus RNase Inhibitor. After separation by FACS, cells were washed, separated into 500,000 cell aliquots, pelleted, snap-frozen and stored at -80°C.
For RNA-seq, RNA libraries were generated using the TruSeq stranded total RNA-seq [Illumina] kit per manufacturer instructions and single-end sequenced for 51 cycles on an Illumina Hi-Seq 2000 or NextSeq 500 per manufacturer instructions. For csRNA-seq, small RNAs of approximately 20–60 nt were size-selected from >1 µg of total RNA with generous spacing on a 15% acrylamide, 7M UREA and 1x TBE gel (Invitrogen EC6885BOX), eluted and precipitated over night at -80°C. Given the RNA fragmentation associated with de-crosslinking, input libraries were generated to eliminate potential false positives and ensure accurate TSS peak calling. csRNA-seq libraries were twice cap selected prior to decapping and libraries were generated as described above. Input libraries were treated with the RppH pyrophosphatase (NEB M0356) prior to adapter ligation to include the whole repertoire of small RNAs with 3’-OH. Samples were quantified by Qbit (Invitrogen). For ChIP-seq, Cells aliquots (500,000) were resuspended on ice in 130 µl RLNR1 lysis buffer (20 mM Tris/HCl pH7.5, 150mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% SDS, 0.4% Na-Deoxycholate, 1% NP-40 alternative, 0.5 mM DTT, 1x protease inhibitor cocktail (DTT and protease inhibitors added immediately before use) and transferred to microtubes with an AFA Fiber (Covaris, MA). All subsequent steps were performed on ice or at 4°C. Chromatin was sheared by sonication in a 96 Place microTUBE Rack (Covaris cat#500282) using a Covaris E220 focused-ultrasonicator (Covaris, MA) for 20 cycles with the following settings: time, 60sec; duty, 5; PIP, 140; cycles, 200; amplitude, 0.0; velocity, 0.0; dwell, 0.0. Samples were recovered and spun at max speed for 10 mins and the pellet was discarded. 1 % of the sample was stored at -20°C for DNA input control and the remaining supernatant was transferred to PCR strips and brought up to a volume of 200 µl using RLNR1 lysis buffer. For ChIP, 20 μl Protein A/G Dynabeads per sample was washed 2x with 1 ml 0.5% BSA/TE/0.1%Tween20 (TET), resuspended in 0.5% BSA/TET, SREBP antibodies (Santa Cruz Biotechnology: sc-8984X, Thermo Fisher Scientific: PA1-337, R&D Systems: AF7119) added, and rotated for 1 hr at RT. The supernatant was removed, Dynabeads washed 1X with 0.1% BSA/TET using a magnet, resuspended in 10 µl RIPA buffer per IP reaction. Dynabeads in RIPA buffer were added to samples and rotated overnight at 4°C. Dynabeads were washed 3 times with Wash Buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), three times with Wash Buffer 3 (10 mM Tris-HCl pH, 250 mM LiCl, 1 mM EDTA, 1% Triton X- 100, 0.7% Na-Deoxycholate) and three times with TET (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.2% Tween20). Dynabeads were washed once with TE-NaCl (10 mM Tris-HCl pH 8, 1 mM EDTA, 50 mM NaCl) and resuspended in 25 μl TT (10 mM Tris-HCl pH 8, 0.05% Tween20). Input samples were resuspended in 25 μl TT and libraries were generated in parallel with ChIP samples. Library NEBNext End Prep and Adaptor Ligation were performed using NEBNext Ultra II DNA Library Prep kit (New England BioLabs) according to manufacturer instructions using barcoded adapters (NextFlex, Bioo Scientific). Libraries were incubated with RNase A and Proteinase K at 55°C for 1 hour and 65°C overnight. Libraries were PCR amplified for 14 cycles with NEBNext High Fidelity 2X PCR MasterMix (New England BioLabs, NEBM0541). Libraries were size selected for 225 – 350 bp fragments by gel extraction (10% TBE gels, Life Technologies).
Samples were sequenced using the Illumina NextSeq 500 platform using 51 cycles (ChIP-seq) or 75 cycles (csRNA-seq) single-end sequencing per manufacturer instructions. RNA-seq samples were single-end sequenced for 51 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA)

treatment_protocol: After monocyte differentiation, on day 7 of culture, moDCs were infected with ZIKV clinical isolate SD001 at MOI 0.5, ZIKV PRVABC59, ZIKV-EGFP-BeH819015 and DENV UIS353 at MOI 1. Briefly, moDCs were incubated 2 hours with the virus, washed with PBS, and incubated in fresh medium for the for 24h at 37˚C, 5% CO2.

tag: donor

value: iPSC Neural progenitor cell

tag: viral infection

value: ZIKV-infected

tag: viral strain

tag: time post infection viral infection


title: KL7_RNAseq_HFD_Het_rep2

description: Sample 13


title: Huh7.5_2_RNAseq_Mock_24h

description: Ribosome-depleted RNA-seq

source_name: Huh7.5 cell line

value: Hepatocyte cell line

value: mock infected


title: Huh7.5_1_RNAseq_ZIKVneg_24h

description: Ribosome-depleted RNA-seq

value: ZIKV-bystander


title: NPC_RNAseq_Mock_24h

description: Ribosome-depleted RNA-seq


title: Huh7.5_2_RNAseq_ZIKVneg_24h

description: Ribosome-depleted RNA-seq


title: exvivo_FT_S1_nonvg9vd2_G, TCR

description:
bulk TCR-seq
FT19_g.csv

value: Gamma delta T cell


title: BALB/c_Rep_3 [175106]

status: Public on Aug 16 2022

first: Christian

last: Büttner

address: Schwabachanlage 10

department: Institut of Human Genetics

institute: University Hospital Erlangen

description: Transcriptome Sequencing

data_processing:
Basecalling with Illumina RTA 1.18.61
Adapter trimming and quality trimming of reads of poly-N-ends and bases with quality lower 20.
Alignment to strain pseudogenomes generated wth GRCm38 Reference using Tophat2 (version 2.1.1) in very high sensitivity mode, the reference being alterted with modtools (version 1.0.2) to include SNPs and short InDels from the Sanger Mouse Genomes Variant call sets (Release 0515).
Conversion of the Tophat2 generated BAM alignments files using Lapels (version 1.1.1) to conform to the standard GRCm38 Reference coordinates again.
Counting of exonic alignments relative to Ensembl Mouse genes model (Release 94) to quantify gene level expression with HTSeq (version 0.7.1) in union mode.
Genome_build: GRCm38.
Supplementary_files_format_and_content: Expression normalized and regularized log-transformed with DESeq2 (version 1.30) function rlog.

source_name: Naive cornea_Balb/c

extract_protocol:
Total RNA was extracted from mouse cornea using the Qiagen RNeasy Micro Kit with DNA digestion.
Nugen Ovation RNA-seq system for model organisms (mouse)

value: Balb/c

value: Naive cornea


title: Huh7.5_2_RNAseq_ZIKVpos_24h

description: Ribosome-depleted RNA-seq


title: NPC_RNAseq_ZIKVneg_24h

description: Ribosome-depleted RNA-seq


title: Huh7.5_1_RNAseq_Mock_24h

description: Ribosome-depleted RNA-seq


title: exvivo_FT_S3_vg9vd2, GEX

description:
bulk RNA-seq
CPM_FT_PNT_Vg9Vd2.csv

extract_protocol:
Cell suspension from FT and PNT samples was obtained as previously described (McGovern et al., 2017; Van Coppernolle et al., 2009). γδ T cells from postnatal suspensions were isolated by positive selection using anti-TCRγδ magnetic activated cell sorting beads (Miltenyi Biotec) according to the manufacturer’s instruction. Thus, the γδ+ fraction was further sorted into CD3+TCRγδ+Vγ9+Vδ2+ subset (all around 10,000 cells) on a FACS Aria III cell sorter (BD Biosciences), snap-frozen in liquid nitrogen, and stored at −80°C for later RNA extraction. The mean purity was 95.0% (percentage of living cells). .RNA was extracted using RNeasy Plus Micro kit according to manufacturer’s instructions.
Library was obtained using the Ovation Solo RNA-Seq System (NuGen) following manufacturer's instruction

value: 17 wk gestation time

value: fetal thymus


title: Huh7.5_1_RNAseq_ZIKVpos_24h

description: Ribosome-depleted RNA-seq


title: KL6_RNAseq_HFD_Het_rep1

description: Sample 12


title: BALB/c_Rep_4 [175107]

description: Transcriptome Sequencing


title: BALB/c_Rep_1 [175101]

description: Transcriptome Sequencing


title: Emb.03-viscera, scRNAseq

status: Public on Aug 15 2022

city: Shanghai

first: Tengjiao

last: Zhang

state: Shanghai

address: 1239 Siping Road

department: School of life science and technology

country: China

institute: Tongji University

description:
10x Genomics
raw_counts.mtx.gz
gene_annotate.csv.gz
cell_annotate.csv.gz

data_processing:
Raw reads were demultiplexed, filtered and mapped to the human reference genome (GRCh38) using Cell Ranger 6.1.2 with default settings. The filtered feature-barcode matrices generated by Cell Ranger were used for further analysis.
To exclude low-quality cells, cells with fewer than 1,000 genes or over the 90th quantile of total UMIs in each library or having higher than 10% UMIs from mitochondrial genome were discarded.
Iterative clustering was performed to identify cell types.
Assembly: GRCh38
Supplementary files format and content: matrix table with cell x gene counts
Supplementary files format and content: csv files with gene and cell annotations
Supplementary files format and content: h5 files with spot x cell counts

molecule: polyA RNA

source_name: embryo viscera

growth_protocol: Aborted embryos from donations were washed with PBS, transferred into cold Hibernate-E medium (Gibco) and immediately transported to the laboratory. Embryos with good quality were staged based on anatomical characters.

extract_protocol:
Samples were incubated at 37°C for 10 minutes and were pipetted up and down thoroughly and gently every 3 minutes. To quench the dissociation, we added 300μl FBS into each tube and centrifuge cells at 300xg for 5 min at 4°C. Cells were then resuspended in 500μl cold PBS, filtered through a 35μm strainer. Single cells were subsequently washed twice with cold PBS, centrifuged at 300xg for 5 min at 4°C and resuspended in cold PBS.
To generate sequencing libraries, a suspension of 10,000 single cells per sample was processed following the 10x Genomics protocol using Single Cell 3’ Reagent Kits v3 (10x Genomics).

treatment_protocol: We dissected each embryo on ice into four main sections, i.e., head, trunk, viscera and limb. After the dissection, each sample was placed into separate tubes, washed twice with cold PBS, cut into pieces and suspended in 300μl Hibernate-E medium with 0.05mg/ml Liberase TM (Roche).

value: embryo viscera

tag: Sex

value: Male

tag: Stage

value: Carnegie Stages 13~14


title: Emb.03-limb, scRNAseq

source_name: embryo limb

value: embryo limb


title: Emb.04-limb, scRNAseq


title: POD 14 [Visium spatial transcriptomics]

status: Public on Aug 12 2022

city: Stanford

first: Michael

last: Januszyk

state: CA

address: 257 Campus Dr

department: Surgery

institute: Stanford University

data_processing:
The libraries were then sequenced using NextSeq (Illumina), and Bcl files were demultiplexed.
Raw FASTQ files and histology images were processed by sample with the Space Ranger software, which uses STAR v.2.5.1b (Dobin et al., 2013) for genome alignment, against the Cell Ranger mm10 reference genome, available at: http://cf.10xgenomics.com/supp/cell-exp/. Raw spaceranger output files for each sample were read into a Seurat class object in R using Seurat’s Load10X in a manner that kept them paired with the low resolution histology images for visualization purposes. This included information such as the number of estimated cell counts, the sum of UMIs per spot, number of expressed genes per spot, and graph-based clustering results. We did not drop any spots given the spatial pattern they presented. Data were normalized using the SCTransform with default parameters. Principal component analysis was performed on the normalized dataset, and the top 30 components were used for neighbor finding, Louvain- based cluster analysis, and UMAP dimension reduction and embedding.
Assembly: mm10

source_name: skin wounds

growth_protocol: Skin tissue from mouse specimens was minced on ice and digested in DMEM-F12 (GIBCO®) with 0.5mg/ml LiberaseTM (Roche) for 30 minutes at 37º in an orbital shaker. The digests were quenched with quench media (DMEM-F12 with 10% Fetal Bovie Serum (FBS, Sigma)), centrifuged at 300 x G for 5 minutes at 4 deg C, resuspended in quench media, filtered through 100, 70, and 40um cell strainers (Falcon cell strainer, ThermoFisher), centrifuged once more, and resuspended in FACS buffer.

extract_protocol:
RNA-seq: The Clontech Smarter Ultra Low Input RNA kit (Takara Bio) was used to generate cDNA from 150 pg total RNA following the manufacturer’s recommendations. Amplified cDNA was purified using SPRI Ampure Beads (Beckman Coulter) and the quality and quantity was measured using a High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer (Agilent Technologies).  cDNA was sheared to an average length of 300 basepairs using a Covaris S2 ultrasonicator (Covaris) and libraries were generated with the Clontech Low Input Library Prep kit (Takara Bio). The samples were uniquely barcoded, pooled, and sequenced on HiSeq (Illumina). scRNA-seq: First strand cDNA synthesis was performed in a 10 ul reaction with 100 Units of Clontech’s Smartscribe reverse transcriptase (Cat. No. 639538), 10 Units RRI, 1X First Strand Buffer (Clontech), 5 mM DTT, 1M Betaine (Sigma B0300-5VL), 6mM MgCl2, 1 uM Template Switch Oligo (TSO, (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′, Exiqon) at 42oC/90 min, 70oC/5 min. PCR pre-amplification was performed in a 25 ul reaction with 1X Kapa HiFi HotStart, 0.1 uM ISPCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′, IDT) at 98oC/3 min, then 25 cycles of 98oC/20 sec, 67 oC/15 sec, 72 oC/6 min, then 72 oC/5 min. Amplified cDNAs were purified by SPRI beads using a Biomek FX automated platform and eluted in 25 ul water, and 2 ul aliquots were run on an Fragment Analyzer High Sensitivity NGS 1-6000 kit for quantitation. Barcoded sequencing libraries were made using the miniaturized Nextera XT protocol of Mora-Castilla et al(43) in a total volume of 4 ul. Pooled libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a NextSeq 500 Illumina platform aiming for 50,000 reads per cell. scATAC-seq: For nuclei isolation, 10x genomics protocol CG000169 Rev D was followed using the low cell input modifications. Transposition, GEM generation and barcoding, post GEM Incubation Cleanup, library construction, and quantification was conducted following the 10x genomics protocol CG000169 Rev D. Pooled libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. Spatial transcriptomics: The Gene Expression Slide & Reagent kit was followed per protocol, and used to produce sequencing libraries.
RNA-seq: The samples were uniquely barcoded, pooled, and sequenced on HiSeq (Illumina). scRNA-seq: cDNA libraries were sequenced on a NextSeq 500 Illumina platform aiming for 50,000 reads per cell. scATAC-seq: cDNA libraries were sequenced on a NextSeq 500 Illumina platform. In total, we generated scATAC-seq profiles from 5,353 cells, which yielded on average 13.0 × 10^3 unique fragments mapping to the nuclear genome. Spatial transcriptomics: The libraries were then sequenced using NextSeq (Illumina).

treatment_protocol: RNA-seq: For mouse fibroblasts, fibroblasts were isolated from fresh specimens using FACS. RNA extraction was performed using Qiagen miRNeasy kit with on column DAnase treatment per the manufacturer’s recommendations. scRNA-seq: Wound fibroblasts were index-sorted into 96-well plates with 4 ul per well of lysis buffer consisting of 1U/ul of Recombinant RNase inhibitor (RRI) (Clontech), 0.1% Triton X-100 (Thermo 85111), 2.5mM dNTP (ThermoFisher), 2.5 uM oligodT30VN (5′AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, IDT). Once sorted, plates were immediately spun down and frozen at -80 deg C. Single cell RNAseq was performed via the method described by Picelli et al (42). Lysis buffer plates were thawed on ice, then heated at 72 oC/3 min in a Biorad C1000 Touch thermal cycler to denature RNA. scATAC-seq:  Single cell ATAC-seq was performed following 10x Genomics protocols. Rainbow mouse wound healing fibroblasts were FACS-isolated as previously described. Spatial Transcriptomics: Wound specimens were rapidly harvested and flash frozen in OCT. Using the Visium Tissue Optimization Slide & Reagent kit, permeabilization time was optimized at a thickness of 10um per section and 37 minutes for mouse cutaneous scar tissue. Wound tissues were cryosectioned at -20 degrees onto gene expression slides.

value: skin wounds

value: unsorted (all cells)

tag: treatment


title: Emb.04-trunk, scRNAseq

source_name: embryo trunk

value: embryo trunk


title: BALB/c_Rep_2 [175102]

description: Transcriptome Sequencing


title: Emb.03-trunk, scRNAseq


title: Emb.04-head, scRNAseq

source_name: embryo head

value: embryo head


title: Emb.04-viscera, scRNAseq


title: POD 0 (unwounded) [Visium spatial transcriptomics]


title: POD 7 [Visium spatial transcriptomics]


title: POD 2 [Visium spatial transcriptomics]


title: K562 e1i  rep2 [ATAC-seq]

city: seattle

first: xueqiu

last: lin

state: wa

address: 1100 Fairview Ave N

institute: FRED HUTCHINSON CANCER CENTER

data_processing:
Bowtie2 was used to map raw FASTQ files to hg19, then paired-end reads were filtered  and converted to non-redundant BEDPE files for each sample.
Assembly: hg19
Supplementary files format and content: non-redundant BEDPE files: each row is one unique paired reads. The columns are: chromosome1, start1, end1, chromosome2, start2, end2, PairedReadID, MAPQ value from Bowtie2, strand1 and strand2.

source_name: dCas9-KRAB expressing K562 cell line

growth_protocol: The K562 (human myelogenous leukemia cells, female, ATCC) cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Waltham, MA) with 10% Tet-system-approved FBS (Life Technologies), GlutaMAX (Thermo Fisher Scientific), and Penicillin-Streptomycin (Thermo Fisher Scientific). K562-KRAB-dCas9 cell line was a gift from Dr. Luke Gilbert (University of California San Francisco) and cultured in the same condition(Gilbert et al., 2014).

extract_protocol: For each ATAC-seq assay, 50,000 cells were collected and washed with PBS. After centrifugation, resuspend cell pellet by gently pipetting up and down 10 times with 100 mL cold Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and then keep on ice for 3 minutes. Add 1 mL Wash Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) to the tube and mix gently. The nuclei were then pelleted by centrifuging at 1000 x g for 10 minutes at 4 °C. Resuspend nuclei pellet with 1 x Transposition Reaction Buffer (50 mM Tris-acetate, pH 7.5, 150 mM potassium acetate, 10mM magnesium acetate, 4 mM spermidine, 0.1% Tween-20, 0.01% Digitonin). Add 5 mL homemade ATAC-seq Tnp complex, then mix well gently and place in a 37 °C thermomixer with 1000 rpm shaking for 30 minutes. After the reaction, DNA was purified with MinElute Reaction Cleanup Kit (QIAGEN, Cat. 28206). Elute DNA with 15 mL elution buffer, then set up PCR reaction by mixing with 2.5 mL each indexed primer (25 mM) and 20 mL NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541S). Run the following PCR program: 72 °C for 5 minutes; 98 °C for 30 seconds; 11 cycles of 98 °C for 10 seconds, 63 °C for 30 seconds, and 72 °C for 1 minute; 72 °C for 5 minutes. Purify 200-650 bp DNA fragments for sequencing.

treatment_protocol: dCas9-KRAB expressing K562 cells were infected with lentiviruses expressing sgRNAs against e3, e7, e3&e7, e1, e4, or e1&e4.

tag: cell line

value: dCas9-KRAB expressing K562 cell line

value: e1 inhibition


title: K562 e4i  rep2 [ATAC-seq]

value: e4 inhibition


title: K562 e7i [ATAC-seq]

value: e7 inhibition


title: K562 e7i  rep2 [ATAC-seq]


title: K562 e3i&e7i [ATAC-seq]

value: e3 and e7 dual inhibition


title: K562 WT rep2_2 [ATAC-seq]

value: WT


title: K562 e3i  rep2 [ATAC-seq]

value: e3 inhibition


title: K562 WT rep2 [ATAC-seq]


title: K562 e4i_H3K27ac ChIP-seq

data_processing:
Bowtie2 was used to map raw FASTQ files to hg19, then paired-end reads were filtered  and converted to non-redundant BEDPE files for each sample.
Assembly: hg19
Supplementary files format and content:  non-redundant BEDPE files: each row is one unique paired reads. The columns are: chromosome1, start1, end1, chromosome2, start2, end2, PairedReadID, MAPQ value from Bowtie2, strand1 and strand2.

extract_protocol: Cells were fixed with 1% Formaldehyde in culture medium at room temperature for 10 minutes. Quench the reaction by adding 250 mM Glycine, then wash cells twice with cold PBS. Three million fixed cells were used for each H3K27ac ChIP-seq assay. Resuspend cells with 1 x TE buffer plus 1 x protease inhibitor cocktail and 1 mM PMSF. Chromatin was sheared by sonication on a Diagenode Bioruptor Pico device at 4 °C for 18 cycles with 30 seconds on and 30 seconds off. Adjust chromatin solution to 1 x RIPA buffer (1 x TE, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100) plus 500 mM NaCl. After centrifuging at 13, 000 rpm for 10 min at 4 °C, chromatin supernatant was collected and 10% of it was saved at input. Mix 2 mg H3K27ac antibody (Abcam, ab4729) with 20 mL Dynabeads Protein A beads (ThermoFischer Scientific, Cat. 10001D), then incubate at room temperature for 1 hour with rotation. Wash beads once with 1 x PBS, then add chromatin solution to beads, and incubate at 4 °C overnight with rotation. Wash beads three times with RIPA buffer plus 500 mM NaCl, then three times with RIPA buffer plus 1 M NaCl, then three times with LiCl buffer (1 x TE, 250 mM LiCl, 0.5% NP-40, 0.5% Sodium Deoxycholate), and finally three times with 1 x TE buffer. Elute DNA and reverse crosslinking with protease K digestion and incubating at 65 °C for over 6 hours. DNA was purified with MinElute Reaction Cleanup Kit (QIAGEN, Cat. 28206). Repair DNA ends with End-It DNA End-Repair Kit (Lucigen, Cat. ER0720), then perform A-tailing with Klenow Fragment (3’ -> 5’ exo-) (NEB, Cat. M0212S) provided with dATP, then followed by adapter ligation with T4 DNA ligase (NEB, Cat. M0202S). Finally, the library was indexed and amplified by PCR, and DNA fragments between 200 bp and 600 bp were purified and sequenced.

tag: chip antibody


title: K562 e1i&e4i_H3K27ac ChIP-seq

value: e1 and e4 dual inhibition


title: K562 WT (For e1 and e4 inhibition experiment)_H3K27ac ChIP-seq


title: K562 e3i&e7i_H3K27ac ChIP-seq


title: K562 e1i_H3K27ac ChIP-seq


title: K562 WT_H3K27ac ChIP-seq


title: K562 e7i_H3K27ac ChIP-seq


title: K562 e3i_H3K27ac ChIP-seq


title: ONB_scSeq

city: Boston

first: Matthew

middle: Joseph

last: Zunitch

state: MA

address: 150 Harrison Ave

institute: Tufts University

data_processing:
Cells were divided between four 10X Chromium lanes for library construction. Libraries were pooled in equimolar ratio and sequenced across two NextSeq550 flowcells.
Single cell gene expression quantification was performed using 10X Cell Ranger v3.0.2
Assembly: GRCh38

source_name: olfactory neuroblastoma

extract_protocol:
Primary biopsy specimen of treatment-naieve ONB was obtained via endoscopic surgical resection and placed in DMEM on ice. Tissue was dissociated with Dispase, followed by Accutase, followed by a brief digestion in trypsin-EDTA. The single cell suspension was cryopreserved at -80 degrees C in DMEM with 10% DMSO.
Constructed using 10X Genomics Single Cell Gene Expression kit following manufacturer's instructions.

value: olfactory neuroblastoma


title: ATAC.09_Parental_0h

status: Public on Aug 11 2022

city: Rotterdam

first: Ning Qing

last: Liu

address: Wytemaweg 80

department: Hematology

country: Netherlands

institute: Erasmus University Medical Center Rotterdam

description: ATAC-seq in E14TG2a (with OsTir1), after 0h of IAA

data_processing:
ATAC-seq data were were mapped against mm10 reference genome using BWA-MEM. The mapped reads with mapping quality score <15, as well as optical PCR duplicates, were discarded using SAMtools. The coverage files were generated using “normalize to 1X genome coverage” methods in deepTools.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files

growth_protocol: E14Tg2a mouse embryonic stem cells (mESCs) and derived cell lines were cultured in serum-free DMEM/F12 (Gibco) and Neurobasal (Gibco) medium (1:1) supplemented with N-2 (Gibco), B-27 (Gibco), BSA (0.05%; Gibco), 104 U of leukemia inhibitory factor (LIF; Millipore), the MEK inhibitor PD0325901 (1 μM; Selleckchem), the GSK-3β inhibitor CHIR99021 (3 μM; Cayman Chemical) and 1-thioglycerol (1.5 × 10−4 M; Sigma-Aldrich) on 0.1% gelatin-coated plates. The cell lines were passaged every two days.

extract_protocol:
ATAC-seq libraries were prepared following a published protocol (Liu et al. 2017). 100 k mouse embryonic stem cells are harvested. Nuclei were isolated, permeabilized, and tagmented using in-house produced Tn5 transposase.
The resulted DNA fragments underwent two sequential 9-cycle PCR amplification and the fragments < 700 bp were purified using SPRI beads (Liu et al., 2017).

treatment_protocol: Protein depletion was induced by addition of auxin (IAA) with a final concentration of 500 μM (I5148-10G, Sigma-Aldrich). Protein degradation was induced at different time points and all samples were collected at the end of the time course.

value: Embyonic stem cell

tag: library type

value: ATAC-seq

tag: genetic modification

tag: time point


title: K562 e1i&e4i  rep2 [ATAC-seq]


title: ATAC.10_Parental_24h

description: ATAC-seq in E14TG2a (with OsTir1), after 24h of IAA

tag: cell type


title: ATAC.06_WAPL_24h

description: ATAC-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 24h of IAA

value: OsTir1; WAPL-AID-eGFP


title: ATAC.08_CTCFWAPL_24h

description: ATAC-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 24h of IAA

value: OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP


title: ATAC.05_WAPL_0h

description: ATAC-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 0h of IAA


title: ATAC.04_RAD21_24h

description: ATAC-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 24h of IAA

value: OsTir1; RAD21-AID-eGFP


title: ATAC.01_CTCF.eGFP_0h

description: ATAC-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 0h of IAA

value: OsTir1; CTCF-AID-eGFP


title: 5mo_organoid iPSC GM07492 18355X1

city: Salt Lake City

first: Yueqi

last: Wang

state: Utah

address: 20 South 2030 East

institute: University of Utah

data_processing:
Reads were processed using the 10X Genomics Cell Ranger 2.0 pipeline and aligned to the human GRCh38 reference assembly (Ensembl v84)
Gene-barcode matrices were constructed for each sample by counting unique molecular identifiers (UMIs).
Assembly: GRCh38
Supplementary files format and content: TSV files and matrix files

source_name: telencephalic organoid

extract_protocol:
Organoids were dissociated into single cell suspension. Single-cell capture, lysis, and cDNA synthesis were performed with the 10x Genomics Chromium at the High Throughput Genomics Core Facility at the University of Utah.
Reverse transcription and library construction were performed using the 10X Chromium Single Cell 3' Reagent Kit.

tag: tissue

value: telencephalic organoid


title: 5mo_organoid EYQ #2 18093X1


title: 5mo_organoid H9 #2 18091X1


title: ATAC.03_RAD21_0h

description: ATAC-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 0h of IAA


title: K562 e3i&e7i  rep2 [ATAC-seq]


title: ATAC.07_CTCFWAPL_0h

description: ATAC-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 0h of IAA


title: 5mo_organoid H9 #1 18054X1


title: RNA.37_Parental_96h_r1

description: RNA-seq in E14TG2a (with OsTir1), after 96h of IAA; re-analysis of GSM3992953

data_processing:
RNA-seq: Raw RNA-seq data were mapped against mm10 reference genome using a TopHat2 pipeline (version 2.1.1) (Kim et al., 2013). The mapped reads with mapping quality score <10 were discarded using SAMtools. The read coverage for each gene in “Mus Musculus GRCm38.92” annotation file was determined using a HTSeq tool (version 0.9.1). The coverage files were generated using “normalize to 1X genome coverage” methods in deepTools. The genes with at least 20 reads in both replicates were kept for further analysis. The filtered expression data were normalized based on the size factors of the individual samples using a DESeq2 package.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files

extract_protocol:
RNA was isolated following a standard TRIzol RNA isolation protocol (Ambion). The cells were lysed using 1 ml of TRIzol reagent, and 200 µl chloroform was added to the lysates. The mixture was vortexed and centrifuged at 12,000 g at 4°C for 15 min. Upper phase was homogenized with 0.5 ml of 100% isopropanol, incubated at room temperature for 10 min, and centrifuged at 4°C for 10 min. The resulted RNA pellet was washed with 75% ice-cold ethanol, dried at room temperature for 10 min, and resuspended in RNase-free water. The isolated RNA was treated with DNase using RNeasy Mini Kit (Qiagen)
RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina).

value: RNA-seq


title: RNA.35_Parental_0h_r1

description: RNA-seq in E14TG2a (with OsTir1), after 0h of IAA; re-analysis of GSM3992951


title: RNA.33_RAD21_24h_r1

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 24h of IAA; re-analysis of GSM3992959


title: RNA.36_Parental_0h_r2

description: RNA-seq in E14TG2a (with OsTir1), after 0h of IAA; re-analysis of GSM3992952


title: RNA.38_Parental_96h_r2

description: RNA-seq in E14TG2a (with OsTir1), after 96h of IAA; re-analysis of GSM3992954


title: RNA.24_WAPL_24h_r2

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 24h of IAA; re-analysis of GSM3992946


title: RNA.30_RAD21_0h_r2

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 0h of IAA; re-analysis of GSM3992956


title: ATAC.02_CTCF.eGFP_24h

description: ATAC-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 24h of IAA


title: RNA.23_WAPL_24h_r1

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 24h of IAA; re-analysis of GSM3992945


title: RNA.28_WAPL_96h_r2

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 96h of IAA; re-analysis of GSM3992950


title: RNA.25_WAPL_48h_r1

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 48h of IAA; re-analysis of GSM3992947


title: RNA.31_RAD21_6h_r1

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 6h of IAA; re-analysis of GSM3992957


title: RNA.29_RAD21_0h_r1

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 0h of IAA; re-analysis of GSM3992955


title: RNA.21_WAPL_6h_r1

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 6h of IAA; re-analysis of GSM3992943


title: RNA.20_WAPL_0h_r2

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 0h of IAA; re-analysis of GSM3992942


title: 5mo_organoid EYQ #1 18072X1


title: RNA.27_WAPL_96h_r1

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 96h of IAA; re-analysis of GSM3992949


title: RNA.18_CTCFWAPL_96h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 96h of IAA


title: Visium_CK_rep4

city: Cambridge

first: Li-Huei

last: Tsai

state: Massachusetts

department: Picower Institute for Learning and Memory

institute: MIT

description:
raw_counts.tsv
annotation_capture_areas.tsv
normalized_counts.tsv
visium_metadata.xlsx

data_processing:
Visium data processing: Samples were processed using Scanpy 1.7.2. The 7 sample data matrices were merged into one matrix which was then processed. The sample id and location of each capture area of the resulting matrix were saved and used for visualization. Counts were normalized (total count of 10,000 per capture area) and logarithmized (using Scanpy’s log1p function). The resulting counts matrix, called raw normalized was used for expression visualization and differential expression analysis. For dimension resolution purposes, the raw normalized matrix was further processed: genes that were not characterized as highly variable enough were filtered out (minimum mean of 0.0125, max mean of 3, minimum dispersion of 0.5), and linear regression was performed to eliminate the effect of covariates (total_counts, and mitochondrial genes percentage). The data was then scaled (standard scaling, max value of 10). Afterwards, PCA was performed, as well as sample-level batch correction, using Harmony. Then, a knn network was constructed for the creation of a UMAP embedding. Clusters were discovered using the Leiden algorithm.
Visium image processing: Immunostaining images were first processed as shades of gray pictures. The largest autofluorescence artifacts were removed manually. The signal was then amplified and cleaned using an 85% contrast increase on each image. As the immunochemistry images of the tissue align perfectly to the pictures taken for 10X Visium purposes, calibration was performed to reconstruct the capture areas grid on the immunostaining images. In that image, for each capture area, the mean signal within a circle maximizing image coverage is calculated, and recorded as the DNA damage signal. After standard scaling of this variable, a threshold of 0.4 standard deviation was set to assign capture areas as positive or negative for DNA damage.
Assembly: mm10
Library strategy: spatial transcriptome

source_name: coronal forebrain brain tissue section

growth_protocol: CK-p25 double transgenic mice were raised and maintained on a doxycycline diet. All mice were induced by removing doxycycline from their diet to drive the expression of p25-GFP in forebrain excitatory neurons. All mice were induced at 3-4 months old.

extract_protocol:
Mice were transcardially perfused with ice-cold saline, then brains were dissected and flash frozen in OCT. A cryostat was used to generate 10μM coronal sections of the hippocampus. These sections were applied to 10X Visium Spatial Gene Expression slides. Sections were immunostained with γH2AX and DAPI following manufacturer’s instructions. Sectioning and staining was performed at the MIT Hope Babette Tang (1983) Histology Core Facility. Sections were imaged immediately after staining using a Olympus FV1200 Laser Scanning Confocal Microscope at the MIT Microscopy Core Facility.
Sections were then used to generate 10X Visium Spatial Gene Expression Libraries according to manufacturer’s instructions at the MIT BioMicro Center.
Libraries were sequenced on a NovaSeq6000 Illumina sequencer according to standard operating procedures.

treatment_protocol: no treatment

tag: strain

value: CK

tag: timepoint


title: RNA.22_WAPL_6h_r2

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 6h of IAA; re-analysis of GSM3992944


title: RNA.16_CTCFWAPL_48h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 48h of IAA


title: RNA.17_CTCFWAPL_96h_r1


title: RNA.26_WAPL_48h_r2

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 48h of IAA; re-analysis of GSM3992948


title: RNA.19_WAPL_0h_r1

description: RNA-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 0h of IAA; re-analysis of GSM3992941


title: RNA.15_CTCFWAPL_48h_r1


title: Visium_CK_rep3


title: RNA.34_RAD21_24h_r2

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 24h of IAA; re-analysis of GSM3992960


title: RNA.14_CTCFWAPL_24h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 24h of IAA


title: Visium_CK_rep1


title: Visium_CKp25_rep4


title: Visium_CKp25_rep2


title: Visium_CK_rep2


title: Term-Seq dPNP_dRho_3

city: State College

first: Paul

last: Babitzke

state: PA

address: 203 Althouse Laboratory

institute: Pennsylvania State University

description: pnpA deletion rho deletion - Termseq

data_processing:
base calling and quality scoring conducted via BaseSpace
adaptor trimming and low quality read removal via Trimmomatic v. 0.38 for RNA-seq .fastq dataset
all reads containing custom oligo were extracted via Cutadapt v. 1.16 for Term-seq .fastq dataset
both RNA-seq and Term-seq .fastq files were mapped to B. subtilis genome using bwa-mem v.0.7.12-r1034
sam -> bam, and bam files split by strand via samtools v. 0.1.19-44428cd
coverage files (.cov) for 3' end mapping and .bedgraph files to illustrate coverage were generated using bedtools v. 2.26.0
2 custom python (python v. 3.8.3) scripts were used to identify 3' ends from Term-seq .fastq derived .cov files (located at: https://github.com/zfmandell/Term-seq)
RNA-seq coverage files generated using bedtools v. 2.26.0
Assembly: Refseq : NC_000964.3
Supplementary files format and content: bedgraph and bigwig files, containing a list of called Term-seq
Library strategy: Term-seq

organism: Bacillus subtilis

source_name: bacterial cell culture

growth_protocol: cells were plated on LB plates at 37°C w/ +2 mM IPTG when required and +20 µg/ml chloramphenicol -> single colonies isolated and grown O/N at 37°C standing culture in +0.4 mM IPTG when required and +20 µg/ml chloramphenicol -> next day, cells were grown at in LB at 37°C +20 µg/ml chloramphenicol

extract_protocol:
Total RNA was extracted from collected cell paste via RNeasy mini-kit (Qiagen, cat # 74104)
total RNA was dephosphorylated using CIP (NEB, discontinued), rRNA was depleted via ribo-zero Gram-positive rRNA depletion kits (Illumina, discontinued) or MICROBExpress (Thermo Fisher), remaining RNA was ligated to custom unique 2', 3’ dideoxy RNA oligonucleotide (IDT), Illumina sequencing libraries were generated from ligation products using TruSeq Stranded mRNA kit (Illumina, cat # 20020594) or NEBNext Ultra Directional (NEB, cat # E7760L)

tag: genotype

value: rpoC-10His pnp::kan rho::tet Cmr Kmr TetR


title: RNA.11_CTCFWAPL_6h_r1

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 6h of IAA


title: Term-Seq dPNP_dRho_2

description: pnpA deletion rho deletion - Termseq


title: RNA.08_CTCF.eGFP_96h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 96h of IAA


title: Visium_CKp25_rep3


title: RNA.06_CTCF.eGFP_24h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 24h of IAA


title: Term-Seq dPNP_2

description: pnpA deletion - Termseq

value: rpoC-10His pnp::kan Cmr Kmr


title: Visium_CKp25_rep1


title: Term-Seq dPNP_dRho_1

description: pnpA deletion rho deletion - Termseq


title: Term-Seq dPNP_3

description: pnpA deletion - Termseq


title: RNA.12_CTCFWAPL_6h_r2


title: RNA.04_CTCF.eGFP_6h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 6h of IAA


title: RNA.05_CTCF.eGFP_24h_r1


title: RNA.09_CTCFWAPL_0h_r1

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 0h of IAA


title: RNA.07_CTCF.eGFP_96h_r1


title: RNA.03_CTCF.eGFP_6h_r1


title: RNA.02_CTCF.eGFP_0h_r2

description: RNA-seq in E14TG2a (with OsTir1; CTCF-AID-eGFP), after 0h of IAA


title: RNA.01_CTCF.eGFP_0h_r1


title: ChIP.14_CTCFWAPL_24h_antiH3K4me1

description: ChIP-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 24h of IAA

data_processing:
Calibrated ChIP-seq data were analyzed based on a method described in previously (Liu et al., 2021). Raw sequencing data was mapped to a concatenated reference genome (mm10 and hg19) using Bowtie 2 mapper (version 2.3.4.1) (Langmead et al., 2009). The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools (version 1.9) (Li et al., 2009). The coverage files (bigWig files) were generated by applying the above computed scaling factor using deepTools (version 2.5.4) (Ramírez et al., 2014), and further calibrated based on average enrichment of the ChIP-seq experiments under direct comparison using their spike-in reference.; Standard ChIP-seq data were mapped to a mm10 reference. The mapped reads with mapping quality score <15 and the optical PCR duplicates were discarded using SAMtools. The coverage files of uncalibrated ChIP-seq data and the data of pluripotency factors (due to absence of these factors in HEK293T cells) were generated using “normalize to 1X genome coverage” methods in deepTools. Peak calling was performed using MACS2 (version 2.1.1.20160309) (Liu, 2014) at a q-value cutoff of 0.01.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files

extract_protocol:
Calibrated ChIP-seq experiments were performed in presence of 10% HEK293T cells as an internal reference using a published protocol (Liu et al., 2021). For chromatin preparation, the mouse embryonic stem cells were mixed with 10% HEK293T cells and cross-linked by a final concentration of 1% formaldehyde for 10 min. The cross-linking reaction was quenched using 2.0 M glycine. The cross-linked cells were then lysed and sonicated to obtain ~300 bp chromatin using Bioruptor Plus sonication device (Diagenode). For ChIP assays, antibodies were first coupled with Protein G beads (ThermoFisher Scientific), and then the sonicated chromatin were incubated overnight at 4oC with the antibody coupled Protein G beads. After over incubation, captured chromatin was washed, eluted and de-crosslinked. The released DNA fragments were purified using MiniElute PCR Purification Kit (Qiagen). The ChIP experiments were performed using the following antibodies: (1) WAPL (16370-1-AP, Proteintech), (2) CTCF (07-729, Merck Millipore), (3) RAD21 (ab154769, Abcam), and (4) H3K4me1 (pAb-037-050, Diagenode).
Calibrated ChIP-seq libraries were prepared according to the protocol of KAPA HTP Library Preparation Kit (Roche); 2. RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina).

value: ChIP-seq


title: ChIP.13_CTCFWAPL_0h_antiH3K4me1

description: ChIP-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 0h of IAA


title: RNA.13_CTCFWAPL_24h_r1


title: RNA.10_CTCFWAPL_0h_r2


title: ChIP.11_CTCFWAPL_24h_antiRAD21


title: ChIP.09_CTCFWAPL_0h_antiRAD21


title: ChIP.10_CTCFWAPL_6h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 6h of IAA


title: ChIP.07_CTCFWAPL_0h_antiCTCF


title: ChIP.06_CTCFWAPL_24h_antiWAPL


title: RNA.32_RAD21_6h_r2

description: RNA-seq in E14TG2a (with OsTir1; RAD21-AID-eGFP), after 6h of IAA; re-analysis of GSM3992958


title: ChIP.08_CTCFWAPL_24h_antiCTCF


title: ChIP.02_WAPL_6h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 6h of IAA


title: ChIP.01_WAPL_0h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 0h of IAA; re-analysis of GSM3992901


title: ChIP.03_WAPL_24h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 24h of IAA; re-analysis of GSM3992902


title: ChIP-Seq-spikein_RPB12_Untreated_rep1

status: Public on Aug 09 2022

city: Beijing

first: Xiong

last: Ji

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

department: School of Life Sciences

institute: Peking University

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB12_Untreated.bw

data_processing:
ChIP-Rx raw data were processed using a custom pipeline that builds on published workflows with minor modifications. Briefly, raw reads after adapter trimming and quality control with cutadapt and RseQC were 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 properly paired and uniquely aligned reads were retained for subsequent processing. Reads 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).
SAMtools (v 0.1.19) and sambamba (v0.7.0) were used to filter unmapped reads, multiple mapped reads, and potential PCR duplicates. The final BAM files were converted to bigwig and normalized to spike-in controls using bamCoverage from deeptools.
For visualization purposes, the bigWig format tracks from two biological replicates for each treatment were merged into an averaged signal using the mean operator from the WiggleTools (v1.2) package (Zerbino, Johnson, et al. 2014).
Assembly: mm10 and dm6
Supplementary files format and content: bigwig files

source_name: mouse embryonic stem cells (V6.5)

growth_protocol: 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. ZBTB11-GFP inducible expressing HEK293T cells were constructed in our lab and used as spike-in cells, which were cultured in basic-DMEM (Thermo Fisher, Cat#C11995500BT) supplemented with 10% FBS (Gibco, Cat#10099141C).

extract_protocol:
Cross-linked cells were subjected to chromatin fraction isolation as reported (Ji et al., 2015), and the chromatin was pretreated with 40 U MNase (NEB, M0247), followed by sonication as previously described (Yongpeng et al., 2020). 1 μl GFP antibodies (Abcam, Ab290) and 30 μl Protein G beads (Thermo Fisher, 10004D) were used to enrich Pol II subunit-DNA complexes.
ChIP DNA were subjected to  library construction according to the instructions of the NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S). 200bp-400bp DNA libraries were gel-selected, purified, and delivered to Novogene company for sequencing.

treatment_protocol: For each RPB subunits ChIP-seq with reference exogenous genome, RPB degron cells were treated with 1μg/ml Dox for 12 h to induce TIR1 expression, and treated with or without 500 μM IAA for 1 h to degrade each RPB subunit. As the spike-in cells, the ZBTB11-GFP was induced by treating ZBTB11-GFP HEK293T cells with 1μg/ml Dox for 24 h. Then RPB degron cells were mixed with 10% ZBTB11-GFP HEK293T cells, and followed with cross-linking.

value: RPB12-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h

tag: antibody


title: ChIP-Seq-spikein_RPB12_IAA_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB12_IAA.bw

value: RPB12-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: Term-Seq dPNP_1

description: pnpA deletion - Termseq


title: ChIP.12_CTCFWAPL_96h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; CTCF-AID-mCherry; WAPL-AID-eGFP), after 96h of IAA


title: ChIP-Seq-spikein_RPB12_IAA_rep2


title: ChIP-Seq-spikein_RPB12_Untreated_rep2


title: ChIP-Seq-spikein_RPB10_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB10_IAA.bw

value: RPB10-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB11_IAA_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB11_IAA.bw

value: RPB11-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB11_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB11_Untreated.bw

value: RPB11-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB10_IAA_rep1


title: ChIP-Seq-spikein_RPB10_Untreated_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB10_Untreated.bw

value: RPB10-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB10_Untreated_rep2


title: ChIP-Seq-spikein_RPB9_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB9_Untreated.bw

value: RPB9-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB9_Untreated_rep1


title: ChIP.05_CTCFWAPL_0h_antiWAPL

value: Embyonic stem cell


title: ChIP-Seq-spikein_RPB8_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB8_IAA.bw

value: RPB8-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP.04_WAPL_96h_antiRAD21

description: ChIP-seq in E14TG2a (with OsTir1; WAPL-AID-eGFP), after 96h of IAA

value: OsTir1; WAPL-AID-eGFP


title: ChIP-Seq-spikein_RPB9_IAA_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB9_IAA.bw

value: RPB9-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB9_IAA_rep2


title: ChIP-Seq-spikein_RPB11_IAA_rep2


title: exvivo_PNT_S3_vg9vd2, GEX

description:
bulk RNA-seq
CPM_FT_PNT_Vg9Vd2.csv

data_processing:
Data processing of single-cell RNA sequencing libraries: Cell Ranger v3.0.2 was used to demultiplex and map sequencing reads against the GRCh38 genome. The resulting count matrices were loaded into R using read10x function form Seurat R package. All downstream analyses were implemented using R v4.0.3 and the package Seurat v3.2.3 (Stuart et al., 2019). Low-quality and doublet cells were filtered. Percentage of mitochondrial genes were plotted as well, and outliers were removed to filter out dead cells. Cell cycle score was calculated for all cells and regressed out. Integration vignette from Seurat v3.0 was followed to generate merged Seurat objects using the SCTransform function and regressing mitochondrial genes and trdv & trgv genes. PCA component was performed using pca Seurat function with 20 dimensions. RunUMAP function was used to obtain bidimensional coordinates for each cell. We determined the k-nearest neighbors of each cell using the FindNeighbors function and used this knn graph to construct the SNN graph by calculating the neighborhood overlap (Jaccard index) between every cell and its k.param nearest neighbors. Finally, we used the FindClusters function to cluster cells using the Louvain algorithm based on the same PCs as RunUMAP function.
Single-cell TCR sequencing analysis: Sc TCR libraries were generated by using CellRanger vdj pipeline (v3.0.2). Human genome GRCh38 was used as reference for alignment. Integrated FT and PNT Seurat objects (gene expression data) were combined with their respective sc TCR-seq (TCR sequence) data based on shared 10× cell barcodes and following the script provided here (https://www.biostars.org/p/384640/). Only those cells expressing productive TCR sequences (γ and/or δ chain) from the ‘filtered_contig_annotations’ file were retained for data integration in the Seurat objects.
RNA-Seq samples: Approximately 30 million paired-end reads (2x101 bases) /sample were collected on a NovaSeq 6000 (Illumina). Paired-end reads with variable trimming (minimum size 36 bases) were mapped against the human reference genome (GRCh38) using STAR_2.5.3a software to generate read alignments for each sample. Annotations Homo_sapiens.GRCh38.90.gtf were obtained from ftp.Ensembl.org. After transcripts assembling, gene level counts were obtained using HTSeq, followed by a library size normalization data processing step. Supplementary_files_format_and_content: CSV file include CPM (count per million) values for each Sample.
Bulk TCR-seq samples: Raw sequencing reads from fastq files (read 1 and read 2) were aligned to reference V, D and J genes from GenBank database specifically for ‘TRG’ or ‘TRD’ to build CDR3 sequences using the MiXCR software version 2.1.1. Default parameters were used except to assemble TRDD gene segment where 3 instead of 5 consecutive nucleotides were applied as assemble parameter. CDR3 sequences were then exported and analyzed using VDJtools software version 1.2.1 using default settings. Sequences out of frame and containing stop codons were excluded from the analysis. Finally data from both files, sorted Vg9Vd2 and nonVg9Vd2, were combined in a same csv file to analysis publicity levels in the single cell dataset.
Assembly: GRCh38
Supplementary files format and content: scRNA-seq: matrix.mtx, features.tsv, barcodes.tsv. // matrix files include transcript count from each single-cell GEX library, barcodes tsv files includes the barcodes present in that single-cell GEX library and features tsv file includes the ID of the genes detected in that single-cell GEX library. /-/ scTCR-seq: TCR_filtered_contig_annotations.csv, TCR_all_contig_annotations.csv. // CSV files include single-cell barcoded CDR3 sequences. /-/ bulk RNA-seq file: CPM_FT_PNT_Vg9Vd2.csv // CSV file include CPM (count per million) transcript values for each sample. /-/ bulk TCR-seq files: FT17_d.csv,FT17_g.csv,FT19_d.csv,FT19_g.csv,FT3_d.csv,FT3_g.csv,PT3_d.csv,PT3_g.csv,PT4_d.csv,PT4_g.csv,PT5_d.csv,PT5_g.csv,PT6_d.csv,PT6_g.csv,PT7_d.csv,PT7_g.csv,PT8_d.csvPT8_g.csv // Each csv file contains CDR3 sequences (gamma for files with a g in the name and delta for files with a d in the name) of sorted gamma delta T cells.

source_name: ϒδ thymocytes

value: Gamma delta T cell

tag: age

value: 1 year and 1 month

value: pediatric thymus


title: ChIP-Seq-spikein_RPB8_Untreated_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB8_Untreated.bw

value: RPB8-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB7_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB7_IAA.bw

value: RPB7-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB11_Untreated_rep1


title: ChIP-Seq-spikein_RPB6_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB6_IAA.bw

value: RPB6-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB6_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB6_Untreated.bw

value: RPB6-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB7_Untreated_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB7_Untreated.bw

value: RPB7-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB6_IAA_rep1


title: ChIP-Seq-spikein_RPB6_Untreated_rep1


title: ChIP-Seq-spikein_RPB5_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB5_IAA.bw

value: RPB5-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB5_IAA_rep1


title: ChIP-Seq-spikein_RPB7_IAA_rep1


title: ChIP-Seq-spikein_RPB5_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB5_Untreated.bw

value: RPB5-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB5_Untreated_rep1


title: ChIP-Seq-spikein_RPB8_Untreated_rep2


title: ChIP-Seq-spikein_RPB4_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB4_IAA.bw

value: RPB4-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: Ribo_6h_KCl_Harringtonin_rep3

first: Erin

middle: Elizabeth

last: Duffy

address: 200 Longwood Ave, Goldenson 405

department: Neurobiology

institute: Harvard Medical School

description: Ribosome-associated 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.
Assembly: hg38
Supplementary files format and content: counts
Library strategy: Ribo-seq

source_name: hESC-derived 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. For harringtonine treatment, 2 μg/mL harringtonine or an equal volume of DMSO was added to cell culture media and incubated at 37°C for 2 min before proceeding with Ribo-seq cell lysis and sample preparation as described above.

value: Cultured Neurons

tag: agent


title: Ribo_6h_KCl_Harringtonin_rep2

description: Ribosome-associated RNA


title: Ribo_6h_KCl_Harringtonin_rep1

description: Ribosome-associated RNA


title: ChIP-Seq-spikein_RPB4_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB4_Untreated.bw

value: RPB4-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB8_IAA_rep1


title: ChIP-Seq-spikein_RPB3_IAA_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB3_IAA.bw

value: RPB3-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: Ribo_6h_KCl_DMSO_rep3

description: Ribosome-associated RNA


title: ChIP-Seq-spikein_RPB4_IAA_rep1


title: Ribo_6h_KCl_DMSO_rep2

description: Ribosome-associated RNA


title: Ribo_6h_KCl_DMSO_rep1

description: Ribosome-associated RNA


title: ChIP-Seq-spikein_RPB3_IAA_rep1


title: ChIP-Seq-spikein_RPB3_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB3_Untreated.bw

value: RPB3-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB3_Untreated_rep1


title: ChIP-Seq-spikein_RPB7_Untreated_rep2


title: ChIP-Seq-spikein_RPB2_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB2_Untreated.bw

value: RPB2-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h


title: ChIP-Seq-spikein_RPB2_IAA_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB2_IAA.bw

value: RPB2-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB1_IAA_rep1

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB1_IAA.bw

value: RPB1-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h, then treated with 500 μM IAA for 1h


title: ChIP-Seq-spikein_RPB1_Untreated_rep2

description:
NEBNext Ultra II DNA Library Prep Kit (NEB, cat#E7645S)
ChIP-Seq-spikein_RPB1_Untreated.bw

value: RPB1-mAID-GFP degron mESCs treated with 1μg/ml Dox  for 12 h