title: JL041 Tcon I CD4 d7


title: JL041 Tcon II CD8 d7

status: Public on Apr 23 2021

type: SRA

city: Stanford

first: Juliane

last: Lohmeyer

address: 269 W. Campus Drive

country: USA

institute: Stanford University

description:
bulkRNA-seq
BulkRNAseq_Tcon_PrePostTx_RawCounts.csv

library_source: transcriptomic

data_processing:
scRNAseq: Sample demultiplexing, barcode processing, alignment to the mm10/GRCm38 reference genome, and single-cell 3′ gene counting were performed using the Cell Ranger Suite version 2.1. scRNA-Seq analysis was performed using the Seurat R package, version 3.
Bulk RNAseq on iNKT sublineages: Paired-end reads were aligned to the mm10 genome (GRCm38 v90) using STAR v. 2.5.4, and genes quantified using HTSEQ24. Differential gene expression was performed using the DESeq2 version 1.22.2. To generate the genome tracks, we used samtools to make stranded BAM files for each library from the SAM file. The BAM file was used to generate a bedGraph using the genomeCoverageBed command. BedGraph from multiple replicates were aggregated using bedtoolsunionbedg, and each track was normalized to the same sequencing depth of 30 million reads. The averaged bedGraph were converted to BigWig files using the command bedGraphToBigWig. Statistics from the mapped reads were aggregated using MultiQC.
ATACseq: Sequencing reads were processed using the ENCODE ATAC-seq pipeline (https://github.com/kundajelab/atac-seq-pipeline) with default parameter to detect and removes adapters, align reads to the specified mm10 reference genome using Bowtie2, and subsequently remove unmapped, mate unmapped, multimapped, mitochondrial mapped, and PCR duplicate reads. Peaks are called on replicates using MACS2. Reads overlapping consensus peaks were quantified for each cell subset replicate using bedtools version 2.27.1. Subset enriched peaks from the consensus set were determined with DESeq2 Consensus peak region annotations with nearest gene were obtained using HOMER version 4.9.1. Gene coordinates for genes-of-interest were queried using biomaRt version 2.34.2 using mmusculus_gene_ensembl for mm10 (GRCm38.6). Transcription factor motifs enrichment were identified using HOMER version 4.9.1.
Bulk RNAseq on Tcon post-transplantation: Sequencing reads were checked using FastQC v.0.11.7. Estimated transcript counts and transcripts per million (TPM) for the mouse genome assembly GRCm38 (mm10) were obtained using the pseudo-aligner Kallisto. Transcript-level abundance was quantified and summarized into gene level using the tximport R package. Differential gene expression was performed using the DESeq2 R package version 1.22.221.
Genome_build: mm10
Supplementary_files_format_and_content: all processed files: csv, raw counts

molecule: total RNA

organism: Mus musculus

source_name: spleen, peripheral lymph nodes

extract_protocol:
Bulk RNA-seq of iNKT sublineages: RNA was extracted from freshly FACS-sorted iNKT sublineages using the RNeasy mini kit (Qiagen) after pelleting 10k-50k cells, ATAC-seq: Lysis was performed in 100 μl cold buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 + 0.1% IGEPAL CA-630 + 0.1% Tween 20), and transposition was performed in 50 μl buffer containing 25 μL 2× TD buffer (Illumina #FC-121-1030), 2.5 μL Tn5 transposase (Illumina #FC-121-1030), 17.5 μL nuclease free H2O, and 5µl 1% digitonin. Bulk RNA-seq of conventional T cells after HCT: RNA was extracted using the TRIzol RNA isolation method (ThermoFisher Scientific) combined with the RNeasy MinElute Cleanup (Qiagen).
Single-cell libraries were generated using the Chromium Controller Single-Cell Instrument and Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics). Bulk cDNA libraries of sorted iNKT sublineages were prepared using the Truseq stranded mRNA kit (Illumina) and pooled for sequencing on the Illumina HiSeq 4000 platform (75 bp, paired-end). ATAC seq was performed as previously described (Buenrostro et al., Nat. Methods 2013), following the reommended library preparation protocol and sequencing on and Illumina HiSeq 4000. Bulk RNA-seq of conventional T cells after HCT: Full-length cDNA was generated using the Clontech SMARTer v4 kit (Takara Bio USA, Inc., Mountain View, CA) prior to library generation with the Nextera XT DNA Library Prep kit (Illumina, Inc., San Diego, CA). Libraries were pooled for sequencing on the Illumina HiSeq 4000 platform (75 bp, paired-end).
RNA-seq, ATAC-seq

tag: cell type

value: conventional T cells

tag: strain

value: FVB/N

tag: treatment

value: GVHD, untreated


title: Lenti_OSM_2_Day7_F08

city: Rehovot

first: Rony

last: Chanoch

state: Israel

address: Weizmann Institute of Science, 234 Herzl Street

country: Israel

institute: Weizmann Instititue

data_processing:
Paired-end reads from mouse model were mapped to the UCSC mm10 mouse transcriptome using Bowtie. Expression values were calculated by RSEM in paired-end mode using parameters, from which TPM values for each gene were extracted. Reads from 10x were aligned using CellRanger
We examined as quality control the number of genes detected in each cell. We observed a bimodal distribution and conservatively excluded sequenced cells with fewer than 3,000 detected genes.
For the remaining cells, we calculated Eavg(i) = log2(mean(TPMi,1...n)+1) for each gene, and excluded genes with Eavg < 4
Genome_build: mm10, hg19
Supplementary_files_format_and_content: Comma-separated value containing TPM values (transcript-per-million reads).

molecule: polyA RNA

source_name: Glioblastoma single cell

extract_protocol:
For mice samples, collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. The dissociated cells were first stained with calcein Blue AM (Thermo Fisher Scientific) and Zombie NIR (BioLegend) for 25 min at 4 ℃, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 1% BSA / PBS, the cells were then stained with a combination of following antibodies; PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences), phycoerythrin (PE) anti-mPdgfra (clone APA5, BioLegend), Allophycocyanin (APC) anti-mPodoplanin (clone 8.1.1, BioLegend), and APC anti-CD11b (clone M1/70, BioLegend), for 30 min at 4 ℃. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive or CD45 positive/ CD11b positive single-cells into 96-well plates containing 5uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at -80 ℃ prior to whole transcriptome amplification, library preparation and sequencing.
Libraries from isolated single cells from mouse model were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications. RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter) before oligo-dT primed reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 768 cells with unique barcodes were combined and sequenced with paired-end, 38-base reads, using a NextSeq 500 sequencer (Illumina). Comma-seperated file containing TPM values (transcript-per-million reads) for 6608 genes across 679 cells. For the cell lines, scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3’ Kit v2 and the 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3’ v2 protocol. The cell lines were processed as a pool that consisted of 6 total cell lines, 4 of which were used in this study (MGG23, MGG75, MGH143, MGG18). Two of the used cell lines, MGG23 and MGH143 were profiled again in a different pool. Briefly, we generated a single cell suspension of the pool in 0.04% PBS-BSA (95% viability) and loaded approximately 10,500 single cells to the Chromium Controller with a targeted recovery of 6,000 cells. Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 12; 72°C for 1 min). The amplified cDNA was fragmented, end-repaired, ligated with index adaptors, and size-selected with clean-ups between each step using the SPRIselect Reagent Kit (Beckman Coulter). Quality control of the resulting barcoded libraries was performed with the Agilent TapeStation and by PCR with primers specific to the P5 and P7 sequence (NEBNext Library Quant Kit for Illumina, New England Biolabs).
single cell RNA-seq


title: JL042 CD4 Tcon d0

value: pre-transplant


title: Lenti_OSM_2_Day7_F06


title: Lenti_OSM_2_Day7_G01


title: Lenti_OSM_2_Day7_F05


title: Lenti_OSM_2_Day7_F04


title: Lenti_OSM_2_Day7_F11


title: JL041CD8 Tcon d0 I


title: JL041 CD4 Tcon d0 II


title: JL041 CD4 Tcon d0 I


title: Lenti_OSM_2_Day7_F03


title: iNKT17-1 ATAC

description:
ATAC-seq
ATACseq_iNKTsublineages_RawCounts.csv

library_source: genomic

molecule: genomic DNA

source_name: Thymus

value: invariant natural killer T cell


title: iNKT17-2 ATAC


title: Lenti_OSM_2_Day7_F02


title: Lenti_OSM_2_Day7_F01


title: Lenti_OSM_2_Day7_E12


title: Lenti_OSM_2_Day7_E11


title: iNKT2-2 ATAC


title: JL041CD8 Tcon d0 II


title: Lenti_OSM_2_Day7_E10


title: Lenti_OSM_2_Day7_E04


title: iNKT1-1 ATAC


title: iNKT2-1 ATAC


title: Lenti_OSM_2_Day7_E06


title: Lenti_OSM_2_Day7_E07


title: iNKT2-1

description:
bulkRNA-seq
BulkRNAseq_iNKTsublineages_RawCounts.csv


title: iNKT17-1


title: iNKT17-2


title: iNKT2-2


title: Lenti_OSM_2_Day7_E02


title: iNKT1 CD4pos 2


title: iNKT1 CD4neg-2


title: Lenti_OSM_2_Day7_E01


title: Lenti_OSM_2_Day7_D11


title: Lenti_OSM_2_Day7_D12


title: iNKT1-2 ATAC


title: Lenti_OSM_2_Day7_D10


title: Lenti_OSM_2_Day7_E08


title: Lenti_OSM_2_Day7_E03


title: iNKT1-1


title: Lenti_OSM_2_Day7_E05


title: iNKT1 CD4neg-1


title: iNKT1 CD4pos 1


title: iNKT1-2


title: Lenti_OSM_2_Day7_D09


title: Control-5

city: INDIANAPOLIS

first: Xiaoyu

last: Lu

state: IN

address: 410W 10TH ST

department: school of medicine

institute: INDIANA UNIVERSITY

description:
5A_Ast-H7-isog-ctr-exp5_S47
merge_counts_matrix.txt

data_processing:
Data is mapping to human genome GRCh38 using STAR software
counts data are extract using featureCounts

organism: Homo sapiens

growth_protocol: hPSCS were differentiated into astrocytes, and then plated on laminin-coated substrates for 3 weeks. RNA was collected directly from the culture dish.

extract_protocol: The astrocytes were lysed and total RNA was extracted  with the PicoPure RNA isolation kit.

tag: genotype

value: isog-ctr


title: Control-4

description:
4A_Ast-H7-isog-ctr-exp4_S45
merge_counts_matrix.txt


title: Lenti_OSM_2_Day7_D06


title: OPTN(E50K)-5

description:
5B_Ast-H7-E50k-exp5_S48
merge_counts_matrix.txt


title: Lenti_OSM_2_Day7_D07


title: Lenti_OSM_2_Day7_D04


title: OPTN(E50K)-4

description:
4B_Ast-H7-E50k-exp4_S46
merge_counts_matrix.txt


title: Lenti_OSM_2_Day7_D02


title: Lenti_OSM_2_Day7_D08


title: Lenti_OSM_2_Day7_D05


title: Lenti_OSM_2_Day7_D03


title: OPTN(E50K)-2

description:
2B_Ast-H7-E50k-exp2_S42
merge_counts_matrix.txt


title: OPTN(E50K)-1

description:
1B_Ast-H7-E50k-exp1_S40
merge_counts_matrix.txt


title: Lenti_OSM_2_Day7_C12


title: Lenti_OSM_2_Day7_D01


title: Lenti_OSM_2_Day7_C09


title: Lenti_OSM_2_Day7_C10


title: AC070_Gingivitis (RNA-seq)

city: Memphis

first: Shannon

last: McKinney-Freeman

state: TN

address: 262 Danny Thomas Place

department: Hematology

institute: St. Jude Children's Research Hospital

data_processing:
RTA (Illumina) was used for base calling and bcl2fastq2 (version 2.17) for converting BCL to fastq format, coupled with adaptor trimming. Then the reads were mapped to a reference genome  using STAR(2.5.2b) and featureCounts(v1.5.0-p3).
Genome_build: hg19
Supplementary_files_format_and_content: raw count text files

source_name: Gingival tissue from gingivitis

extract_protocol:
gingival tissue samples were harvested in conjunction with an invasive oral surgical procedure required for the participant’s oral care after administration of local anesthesia.
Poly-A pull-down was carried out to enrich mRNAs from total RNA samples (200ng-1g per sample) followed by library preparation using the Illumina TruSeq RNA prep kit.

tag: tissue

value: Gingival tissue from gingivitis

tag: Sex

value: M

tag: age

tag: race/ethnicity

value: Hispanic/Latino

tag: batch

value: B

tag: phenotype

value: Gingivitis


title: Control-2

description:
2A_Ast-H7-isog-ctr-exp2_S41
merge_counts_matrix.txt


title: AC068_Gingivitis (RNA-seq)

value: F

value: White


title: AC062_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_C08


title: AC060_Healthy (RNA-seq)

source_name: Healthy gingival tissue

value: Healthy gingival tissue

value: Healthy


title: Control-1

description:
1A_Ast-H7-isog-ctr-exp1_S39
merge_counts_matrix.txt


title: AC072_Gingivitis (RNA-seq)

value: Arfican-American


title: AC066_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_C06


title: AC058_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_C05


title: Lenti_OSM_2_Day7_C04


title: Lenti_OSM_2_Day7_C02


title: Lenti_OSM_2_Day7_C01


title: AC052_Healthy (RNA-seq)


title: AC054_Healthy (RNA-seq)

value: Asian


title: AC050_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_C03


title: AC048_Periodontitis (RNA-seq)

source_name: Gingival tissue from periodontitis

value: Gingival tissue from periodontitis

value: Periodontitis


title: AC046_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_B11


title: AC044_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_B12


title: AC064_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_B09


title: Lenti_OSM_2_Day7_C11


title: Lenti_OSM_2_Day7_B05


title: AC056_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_B08


title: Lenti_OSM_2_Day7_B07


title: AC042_Periodontitis (RNA-seq)


title: AC039_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_B01


title: AC037_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_C07


title: Lenti_OSM_2_Day7_B10


title: AC024_Healthy (RNA-seq)

value: A


title: AC034_Gingivitis (RNA-seq)


title: AC030_Gingivitis (RNA-seq)


title: AC022_Healthy (RNA-seq)


title: AC032_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_B04


title: Lenti_OSM_2_Day7_B06


title: Lenti_OSM_2_Day7_A12


title: AC035_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_A11


title: Lenti_OSM_2_Day7_B02


title: AC028_Gingivitis (RNA-seq)


title: AC019_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_A10


title: AC018_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_B03


title: AC026_Gingivitis (RNA-seq)


title: Lenti_OSM_2_Day7_A09


title: AC013_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_A08


title: AC011_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_A07


title: Lenti_OSM_2_Day7_A05


title: AC016_Healthy (RNA-seq)


title: Lenti_OSM_2_Day7_A06


title: AC005_Periodontitis (RNA-seq)


title: AC003_Periodontitis (RNA-seq)


title: AC007_Periodontitis (RNA-seq)


title: Lenti_OSM_2_Day7_A03


title: AC001_Periodontitis (RNA-seq)


title: L1723-In_vitro_dCas9_Nanog2_mouse

first: Georgi

middle: Kolev

last: Marinov

state: California

address: 279 Campus Drive West, Beckman Center, B-257A/259

department: Genetics

institute: STANFORD UNIVERSITY

data_processing:
read alignment, ChIP-seq data: Bowtie 1.0.1
bigWig file generation: custom code
genome build: hg38
Supplementary_files_format_and_content: bedGraph of read coverage, normalized to RPM

extract_protocol:
In vitro CasKAS experiments were executed as follows. First, 1 uL of each synthetic sgRNA were incubated at room temperature with 1 uL of recombinant purified dCas9 (MCLab dCAS9B-200) for 20 minutes. The RNP was then incubated with 1 ug of gDNA at 37C for 10 minutes.  The KAS reaction was then carried out by adding 1 $\mu$L of 500 mM N$_3$-kethoxal (ApeXBio A8793). DNA was immediately purified using the MinElute PCR Purification Kit (Qiagen 28006), and eluted in 87.5 or 175 uL 25mM K3BO3.
The click reaction was carried out by combining 175 uL purified and sheared DNA, 5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 20 uL 10x PBS in a final volume of 200 uL or 87.5 uL purified and sheared DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 uL 10x PBS in a final volume of 100 uL. The reaction was incubated at 37C for 90 minutes. DNA was purified using AMPure XP beads (50 uL for a 100 uL reaction or 100 uL for a 200 uL reaction), beads were washed on a magnetic stand twice with 80\% EtOH, and eluted in 130 uL 25mM K3BO3. Purified DNA was then sheared on a Covaris E220 instrument down to ~150-400 bp size. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05\% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes (in a Thermomixer, with shaking at 1,000 rpm) and then at 65C for 30 minutes. Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes (in a Thermomixer, with shaking at 1,000 rpm) Beads were then separated on a magnetic stand, and washed with 300 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1X TE buffer, then resuspended in 15 uL 0.1X TE buffer, and heated at 98C for 10 minutes. For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2X NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes. Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8X AMPure XP beads. Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (2x36 cycles).

tag: chip-antibody


title: L1724-In_vitro_Cas9_Nanog3_mouse

value: Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602)


title: Lenti_OSM_2_Day7_A04


title: L1722-In_vitro_Cas9_Nanog2_mouse


title: Lenti_OSM_2_Day7_A02


title: AC009_Periodontitis (RNA-seq)

value: Indian


title: Lenti_OSM_1_Day7_H11


title: L1428-In_vitro_Cas9_sgRNA_2_mouse


title: Lenti_OSM_1_Day7_H10


title: Lenti_OSM_1_Day7_H08


title: L1725-In_vitro_dCas9_Nanog3_mouse


title: Lenti_OSM_1_Day7_H07


title: L1412-In_vitro_dCas9_sgRNA_2_mouse


title: L1427-In_vitro_Cas9_sgRNA_1_mouse


title: Lenti_OSM_2_Day7_A01


title: Lenti_OSM_1_Day7_H12


title: L1411-In_vitro_dCas9_sgRNA_1_mouse


title: Lenti_OSM_1_Day7_H06


title: Lenti_OSM_1_Day7_H09


title: L1239-In_vitro_dCas9_sgRNA_2_mouse


title: L1238-In_vitro_dCas9_sgRNA_1_mouse


title: Lenti_OSM_1_Day7_H05


title: Lenti_OSM_1_Day7_H04


title: Lenti_OSM_1_Day7_H03


title: Lenti_OSM_1_Day7_G10


title: Lenti_OSM_1_Day7_H02


title: Lenti_OSM_1_Day7_G11


title: Lenti_OSM_1_Day7_H01


title: Lenti_OSM_1_Day7_G08


title: Lenti_OSM_1_Day7_G09


title: 00078A2595_VoLo

city: La Jolla

first: Daniel

last: Munro

state: CA

address: 9500 Gilman Drive

department: Psychiatry

institute: UC San Diego

data_processing:
Reads were first trimmed for adapter and poor-quality base calls using cutadapt
Reads were then aligned to the Ensembl Rat Transcriptome using RSEM
Upper quantile adjusted was applied to estimated gene read counts using DESeq2
Samples were filtered based on low reads counts, mismatched genotypes, and expression PCA outliers
Genes were eliminated if less than 25% of libraries had more than one read or if the total number of reads among all libraries for the gene was less than 100
Read counts were log base 2 transformed after the addition of 1 to each read count (to avoid taking the log of zero)
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: Tab-separated table with row names (Ensembl gene IDs) and column names (sample IDs) containing log2(read count + 1) values

organism: Rattus norvegicus

source_name: Brain region: Orbitofrontal cortex

growth_protocol: All rats were group housed under standard laboratory conditions.

extract_protocol:
1) Brain region dissection: Brains were taken out of a minus 80 degree freezer and cryosectioned into 60um sections, which were mounted onto RNase-free glass slides. Slides were stored in minus 80 degree until dissection. During dissection, slides were placed on a minus 20 degree cold plate. One drop (approximately 50 ul) of RNAlater was placed on the brain region of interest. Each brain region then was dissected out under a dissecting video camera by using a pair of fine tipped forceps with the assistance of an 18 gauge needle with a bent tip. Bilateral tissue of the same brain region from each rat was immediately transferred into 350 ul Buffer RLT (containing beta-mercaptoethanol) and placed on dry ice. Tissue was stored in minus 80 degree before RNA extraction. 2) RNA extraction: Tissue was thawed on ice and homogenized by using a clean stainless steel bead using Qiagen TissueLyser (40Hz, 3 min). AllPrep DNA/RNA mini kit (Qiagen) was used to extract RNA. Samples were processed by using the QIAcube robot following standard protocols. The optional DNase digestion step was included for RNA samples.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: All rats were naïve to behavioral or pharmacology treatment.

tag: population

value: Heterogeneous Stock

tag: rat batch

tag: brain region

value: Orbitofrontal cortex


title: 00078A2496_VoLo


title: 00078A22DF_VoLo


title: 00078A2463_VoLo

value: M


title: 00078A2315_VoLo


title: 00078A261F_VoLo


title: Lenti_OSM_1_Day7_G07


title: 00078A2667_VoLo

description: Removed: genotype mismatch


title: 00078A1F34_VoLo


title: Lenti_OSM_1_Day7_G06


title: 00078A1B05_VoLo


title: 00078A1A2B_VoLo


title: 00078A1A16_VoLo


title: Lenti_OSM_1_Day7_G05


title: Lenti_OSM_1_Day7_G12


title: Lenti_OSM_1_Day7_G04


title: Lenti_OSM_1_Day7_G02


title: Lenti_OSM_1_Day7_G03


title: 00078A22EB_VoLo


title: 00078A19D6_VoLo


title: Lenti_OSM_1_Day7_G01


title: Lenti_OSM_1_Day7_F12


title: 00078A19A7_VoLo


title: 00078A19B7_VoLo


title: 00078A19B5_VoLo


title: 00078A19C0_VoLo


title: Lenti_OSM_1_Day7_F11


title: Lenti_OSM_1_Day7_F09


title: Lenti_OSM_1_Day7_F10


title: Lenti_OSM_1_Day7_F06


title: Lenti_OSM_1_Day7_F07


title: Lenti_OSM_1_Day7_F05


title: 00078A194B_VoLo


title: Lenti_OSM_1_Day7_F08


title: 00078A193E_VoLo

description: Removed: low read count


title: Lenti_OSM_1_Day7_F04


title: 00078A1979_VoLo


title: 00078A1905_VoLo


title: 00078A1937_VoLo


title: 00078A18CF_VoLo


title: Lenti_OSM_1_Day7_F02


title: 00078A18A7_VoLo

description: Removed: genotype mismatch


title: 00078A189F_VoLo


title: 00078A1942_VoLo


title: 00078A1875_VoLo


title: 00078A192C_VoLo


title: 00078A1863_VoLo


title: 00078A1837_VoLo


title: Lenti_OSM_1_Day7_E10


title: Lenti_OSM_1_Day7_E08


title: Lenti_OSM_1_Day7_E07


title: 00078A181B_VoLo


title: Lenti_OSM_1_Day7_F03


title: 00078A17A7_VoLo


title: 00078A17F7_VoLo


title: Lenti_OSM_1_Day7_E04


title: Lenti_OSM_1_Day7_E03


title: 00078A18F7_VoLo


title: Lenti_OSM_1_Day7_E12


title: Lenti_OSM_1_Day7_F01


title: Lenti_OSM_1_Day7_E11


title: 00078A186C_VoLo


title: Lenti_OSM_1_Day7_E09


title: Lenti_OSM_1_Day7_E06


title: 00078A1816_VoLo


title: Lenti_OSM_1_Day7_E05


title: 00078A1807_VoLo


title: 00078A0A43_VoLo


title: 00078A16D3_VoLo


title: 00078A1731_VoLo


title: 00078A0AEA_VoLo


title: 00078A179C_VoLo


title: 00078A1732_VoLo


title: 00078A1707_VoLo


title: 00078A087B_VoLo


title: 00078A09B1_VoLo


title: 00078A07A2_VoLo


title: 00078A1772_VoLo


title: 00078A096C_VoLo

description: Removed: low read count


title: 00078A02DF_VoLo


title: 00078A16DF_VoLo


title: 00078A0255_VoLo


title: 00078A0244_VoLo


title: 00078A02CB_VoLo


title: 00078A0246_VoLo

description: Removed: low read count


title: 00078A0224_VoLo


title: 00078A01FE_VoLo


title: 00078A021A_VoLo


title: 00078A0215_VoLo


title: 00078A022D_VoLo


title: 00078A01C0_VoLo


title: 00078A01DB_VoLo


title: 00078A01D8_VoLo


title: Lenti_OSM_1_Day7_E02


title: Lenti_OSM_1_Day7_E01


title: Lenti_OSM_1_Day7_D10


title: Lenti_OSM_1_Day7_D11


title: Lenti_OSM_1_Day7_D09


title: Lenti_OSM_1_Day7_D12


title: Lenti_OSM_1_Day7_D07


title: Lenti_OSM_1_Day7_C05


title: Lenti_OSM_1_Day7_C10


title: Lenti_OSM_1_Day7_C07


title: Lenti_OSM_1_Day7_C11


title: Lenti_OSM_1_Day7_C03


title: Lenti_OSM_1_Day7_D08


title: Lenti_OSM_1_Day7_D03


title: Lenti_OSM_1_Day7_C08


title: Lenti_OSM_1_Day7_C09


title: Lenti_OSM_1_Day7_D05


title: Lenti_OSM_1_Day7_C12


title: Lenti_OSM_1_Day7_C02


title: Lenti_OSM_1_Day7_D01


title: Lenti_OSM_1_Day7_D02


title: Lenti_OSM_1_Day7_C04


title: Lenti_OSM_1_Day7_C06


title: 00078A0189_VoLo


title: Lenti_OSM_1_Day7_D06


title: Lenti_OSM_1_Day7_C01


title: Lenti_OSM_1_Day7_B12


title: 00078A01A6_VoLo


title: Lenti_OSM_1_Day7_D04


title: 00078A0166_VoLo


title: 00078A0139_VoLo


title: 00078A0085_VoLo


title: 00078A00BF_VoLo

description: Removed: low read count


title: 00078A0127_VoLo


title: 00078A0138_VoLo


title: 000789FFF9_VoLo


title: 00078A00AC_VoLo


title: 00078A0058_VoLo


title: 000789FFD3_VoLo


title: 000789FFAC_VoLo


title: 00078A0041_VoLo


title: 00078A002C_VoLo

description: Removed: expression outlier


title: Lenti_OSM_1_Day7_B05


title: Lenti_OSM_1_Day7_B03


title: Lenti_OSM_1_Day7_B10


title: Lenti_OSM_1_Day7_B04


title: Lenti_OSM_1_Day7_B02


title: Lenti_OSM_1_Day7_B07


title: Lenti_OSM_1_Day7_B06


title: Lenti_OSM_1_Day7_B11


title: Lenti_OSM_1_Day7_B08


title: Lenti_OSM_1_Day7_B09


title: Lenti_OSM_1_Day7_B01


title: Lenti_OSM_1_Day7_A12


title: Lenti_OSM_1_Day7_A07


title: Lenti_OSM_1_Day7_A06


title: 000789FFF8_VoLo


title: 000789FF7D_VoLo


title: Lenti_OSM_1_Day7_A05


title: Lenti_OSM_1_Day7_A04


title: Lenti_OSM_1_Day7_A10


title: 000789FFF0_VoLo


title: Lenti_OSM_1_Day7_A08


title: Lenti_OSM_1_Day7_A09


title: Lenti_OSM_1_Day7_A11


title: 000789FF94_VoLo


title: Lenti_OSM_1_Day7_A02


title: Lenti_OSM_1_Day7_A01


title: Lenti_Control_2_Day7_H07


title: 000789FF6E_VoLo


title: 00078A261F_PL

source_name: Brain region: Prelimbic cortex

value: Prelimbic cortex


title: Lenti_OSM_1_Day7_A03


title: Lenti_Control_2_Day7_H08


title: 00077E8336_VoLo


title: 00077E76FE_VoLo


title: Lenti_Control_2_Day7_H06


title: 00077E76C6_VoLo

description: Removed: expression outlier


title: 00078A2463_PL


title: Lenti_Control_2_Day7_H12


title: 00077E67B5_VoLo


title: 00078A1F34_PL


title: 00078A2667_PL

description: Removed: genotype mismatch


title: Lenti_Control_2_Day7_H10


title: 00078A2595_PL


title: Lenti_Control_2_Day7_H09


title: 00078A2496_PL


title: 00078A2315_PL


title: Lenti_Control_2_Day7_H05


title: Lenti_Control_2_Day7_G12


title: Lenti_Control_2_Day7_G11


title: Lenti_Control_2_Day7_G09


title: Lenti_Control_2_Day7_G08


title: Lenti_Control_2_Day7_H04


title: 00078A1A16_PL


title: 00078A19C0_PL


title: 00077EA7E6_VoLo


title: 00078A19A7_PL


title: Lenti_Control_2_Day7_H03


title: 00078A22DF_PL


title: Lenti_Control_2_Day7_G06


title: Lenti_Control_2_Day7_H02


title: 00078A1B05_PL


title: Lenti_Control_2_Day7_G05


title: 00078A194B_PL


title: 00078A1942_PL


title: Lenti_Control_2_Day7_H11


title: Lenti_Control_2_Day7_H01


title: 00078A1A2B_PL


title: 00078A19D6_PL


title: 00078A1905_PL

description: Removed: low read count


title: 00078A192C_PL


title: Lenti_Control_2_Day7_G10


title: Lenti_Control_2_Day7_F11


title: 00078A22EB_PL


title: 00078A1979_PL


title: 00078A18F7_PL


title: 00078A19B7_PL


title: 00078A19B5_PL


title: Lenti_Control_2_Day7_G07


title: Lenti_Control_2_Day7_F07


title: Lenti_Control_2_Day7_F09


title: 00078A186C_PL


title: 00078A193E_PL


title: Lenti_Control_2_Day7_G04


title: Lenti_Control_2_Day7_G03


title: Lenti_Control_2_Day7_G02


title: 00078A1937_PL


title: Lenti_Control_2_Day7_F12


title: Lenti_Control_2_Day7_G01


title: 00078A18CF_PL


title: Lenti_Control_2_Day7_F04


title: Lenti_Control_2_Day7_F10


title: 00078A18A7_PL

description: Removed: genotype mismatch


title: Lenti_Control_2_Day7_F08


title: 00078A1875_PL


title: 00078A179C_PL


title: Lenti_Control_2_Day7_E12


title: Lenti_Control_2_Day7_F05


title: Lenti_Control_2_Day7_F06


title: 00078A1837_PL


title: 00078A1863_PL


title: 00078A181B_PL


title: Lenti_Control_2_Day7_F03


title: 00078A1732_PL


title: Lenti_Control_2_Day7_F02


title: Lenti_Control_2_Day7_F01


title: 00078A1731_PL


title: 00078A17F7_PL


title: 00078A1807_PL


title: Lenti_Control_2_Day7_E06


title: Lenti_Control_2_Day7_E07


title: Lenti_Control_2_Day7_E11


title: Lenti_Control_2_Day7_E09


title: Lenti_Control_2_Day7_E08


title: 00078A1772_PL


title: 00078A1816_PL


title: 00078A17A7_PL

description: Removed: expression outlier


title: 00078A1707_PL


title: 00078A0A43_PL


title: Lenti_Control_2_Day7_E10


title: 00078A09B1_PL


title: 00078A0AEA_PL


title: Lenti_Control_2_Day7_D12


title: Lenti_Control_2_Day7_D11


title: 00078A02DF_PL


title: 00078A16DF_PL


title: Lenti_Control_2_Day7_E05


title: 00078A0246_PL


title: Lenti_Control_2_Day7_E04


title: Lenti_Control_2_Day7_E03


title: 00078A16D3_PL


title: Lenti_Control_2_Day7_D06


title: 00078A096C_PL


title: Lenti_Control_2_Day7_E01


title: 00078A07A2_PL


title: 00078A087B_PL


title: 00078A0224_PL


title: 00078A021A_PL


title: Lenti_Control_2_Day7_D03


title: Lenti_Control_2_Day7_D10


title: Lenti_Control_2_Day7_D04


title: 00078A01FE_PL


title: 00078A02CB_PL


title: Lenti_Control_2_Day7_D02


title: Lenti_Control_2_Day7_D08


title: 00078A01DB_PL


title: 00078A0255_PL


title: 00078A01D8_PL


title: Lenti_Control_2_Day7_C11


title: 00078A01C0_PL


title: 00078A022D_PL


title: Lenti_Control_2_Day7_D09


title: Lenti_Control_2_Day7_D05


title: Lenti_Control_2_Day7_E02


title: 00078A0215_PL


title: 00078A01A6_PL


title: Lenti_Control_2_Day7_C09


title: Lenti_Control_2_Day7_C08


title: 00078A0166_PL


title: In-line-seq

first: Rhiju

last: Das

address: 279 Campus Dr, B419 Beckman Center

department: Biochemistry

institute: Stanford University School of Medicin

data_processing:
Library strategy: Barcode amplicon sequencing
Sequencing reads were analyzed using MAPseeker (https://ribokit.github.io/MAPseeker) following the recommended steps for sequence assignment, peak fitting, background subtraction of the no-modification control, correction for signal attenuation, and reactivity profile normalization.
processed data files format and content: Information about the degradation rates and reactivity rates of each nucleotide in each construct in the library are provided in RDAT format (https://rmdb.stanford.edu/deposit/specs/), a standardized format for reporting construct sequences, experimental conditions, and per-nucleotide signal for nucleic acid probing experiments.

organism: synthetic construct

source_name: in vitro transcribed

extract_protocol:
Quenched RNA samples were purified with 2 µL of Oligo dT bead (Thermo Fisher, AM1922) and 0.8 µL of 10 µM FAM-A20-Tail2. RNA, bead, and primer were incubated at room temperature for 15 min, pulled down by magnetic stand for 10 min, and washed twice with 70% ethanol and left to dry on the bead. RNA was eluted in 2.5 µL of nuclease-free water (RNA was binded with OligodT bead and FAM.A20 Tail2 at this point)
5 µL of purified RNA was added to a reaction mixture containing 1x First Strand buffer (Thermo Fisher), 5 mM dithiothreitol (DTT), 0.8 mM dNTPs, 0.6 µL of SuperScript III RTase (Thermo Fisher) to a final volume of 15 µL. The reaction was incubated at 48 °C for 40 minutes and stopped with 5 µL of 0.4 M sodium hydroxide. The reaction was then incubated at 90 C for 3 minutes, cooled on ice for 3 minutes, and neutralized with 2 µL of quench mix (prepared as 2 mL of 5 M sodium chloride, 3 mL of 3 M sodium acetate, 2 mL of 2 M hydrochloric acid). cDNA was pooled down with 1.5 uL of Oligo C’ beads64 (in house magnetic beads prepared by immobilizing 2x Biotin oligonucleotides with Dynabeads™ MyOne™ Streptavidin C1; Thermo Fisher Scientific 65001), washed twice with 70% ethanol, then resuspended in 3.0 µL of water. We pooled 1.5 µL of each sample together, and took 9 µL of cDNA to continue to ligation an Illumina adapter by using Circ. Ligase I (Lucigen) at 68°C for 2 h. The reaction was stopped by incubation at 80 °C for 10 min. cDNA was added to 10 ul of 5 M NaCl and pulled down with a magnetic stand and washed with 70% ethanol; the ligated product was resuspended in 15 µL H2O.

value: in vitro transcribed


title: Lenti_Control_2_Day7_D01


title: Lenti_Control_2_Day7_C07


title: Lenti_Control_2_Day7_C06


title: 00078A0139_PL


title: 00078A0189_PL


title: 00078A0138_PL


title: Lenti_Control_2_Day7_C05


title: Lenti_Control_2_Day7_C12


title: Lenti_Control_2_Day7_C04


title: 00078A0127_PL


title: Lenti_Control_2_Day7_D07


title: 6d saha rep2

type: RNA

city: Heidelberg

first: Johannes

last: Ridinger

address: Im Neuenheimer Feld 280

department: CCU Pediatric Oncology

country: Germany

institute: German Cancer Research Center

description:
replicate 1
38_6d_SAHA_R2_5617835015

hyb_protocol: Standard Illumina hybridization protocol

scan_protocol: The standard Illumina scanning protocol was used

data_processing: The data were quantile normalized using Illumina BeadStudio Data Analysis Software version v4_r2

label: biotin

source_name: human neuroblastoma cell line SK-N-BE(2)-C

label_protocol: 1mcg total RNA per sample was amplified and biotin labeled using Invitrogen Illumina total Prep reagents, following the standard protocol

growth_protocol: SK-N-BE(2)-C cells were grown in DMEM 4.5g/l glucose supplemented with 10 % FCS and 1 % non-essential amino acids.

extract_protocol: Total RNA was isolated with QIAGEN RNeasy Mini Kit according to manufacturer's instructions

treatment_protocol: Cells were treated with 500 nM vorinostat or vehicle (dmso) for indicated time points

value: vorinostat 144 h

tag: cell line


title: 6d dmso rep2

description:
replicate 2
41_6d_DMSO_R2_5617835015

value: DMSO 144 h


title: Lenti_Control_2_Day7_C10


title: 6d dmso rep3

description:
replicate 1
42_6d_DMSO_R3_5617835015


title: 00078A00AC_PL


title: 24h saha rep3

description:
replicate 3
24_24h_SAHA_R3_5617835006

value: vorinostat 24 h


title: 00078A0085_PL

value: F


title: 24h saha rep2

description:
replicate 2
23_24h_SAHA_R2_5617835006


title: 6d saha rep3

description:
replicate 3
39_6d_SAHA_R3_5617835015


title: Lenti_Control_2_Day7_C02


title: 6d dmso rep1

description:
replicate 1
40_6d_DMSO_R1_5617835015


title: Lenti_Control_2_Day7_C01


title: 24h dmso rep3

description:
replicate 3
30_24h_DMSO_R3_5617835004

value: DMSO 24 h


title: Lenti_Control_2_Day7_B12


title: Yeast_rRNA_H202translated.rep2

city: Barcelona

first: Eva Maria

last: Novoa

address: Dr. Aiguader 88

country: Spain

institute: Center for Genomic Regulation

description: Sample 24

data_processing:
Basecalling with Guppy 3.0.3
Demultiplexing with Deeplexicon
Alignment with Graphmap Default settings
Epinano performed to extract base-called features per position/per 5mer
Nanopolish performed to extract aligned current intensities and subsequently, mean values per position were calculated using a python script
Genome_build: Saccharomyces cerevisiae S288c ribosomal RNA sequences
Supplementary_files_format_and_content: Epinano outputs contain base-called feautures (base quality, mismatch, insertion, deletion frequencies) for each position. Nanopolish outputs contain current intensity mean values for each position

organism: Saccharomyces cerevisiae

growth_protocol: Saccharomyces cerevisiae strains were grown at 30 ˚C in standard YPD medium (1% yeast extract, 2% Bacto Peptone and 2% dextrose). The deletion strains snR3Δ, snR34Δ and snR36Δ were generated on the background of the BY4741 strain by replacing the genomic snoRNA sequence with a kanMX4 cassette as detailed in Parker et al. [57]. For the analysis of rRNAs modifications across polysomal fractions, yeast BY4741 starter cultures were grown in 6 mL YPD medium at 30 ºC with shaking (250 rpm) overnight. 100 mL of fresh YPD medium was inoculated with 10 µL of the stationary culture in a 250 mL erlenmeyer flask, in biological duplicates. Cells were incubated at 30 ºC with shaking (250 rpm) until the cultures reached mid-exponential growth phase (O.D660.~ 0.5-1.0). Yeast cells were then treated with 1 mM H202 or left without treatment (control) for 30 min. 1 mL of cycloheximide stock solution (10 mg/mL) was added to each culture. After 5 min incubation, cells were then quickly transferred into 50 mL pre-chilled falcon tubes, and centrifuged for 5 min at 3,000 g in a 4 ºC pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen.

extract_protocol:
For the samples 1-8 and 17-18-21-22:Saccharomyces cerevisiae BY4741 cells (strains: snR3Δ, snR34Δ snR36Δ, and WT) were harvested via centrifugation at 3000 RPM for 1 minute, followed by two washes with water. RNA was purified from pelleted cells using a MasterPure Yeast RNA extraction kit (Lucigen, MPY03100), according to manufacturer’s instructions., For the rest (Fractions): Yeast pellets from 100 mL cultures were washed with 6 mL of ice-cold Polysome Extraction Buffer (PEB), which contained 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 100 U/mL RNAse inhibitors (RNaseOUT, Invitrogen, #18080051). Cells were centrifuged for 5 min at 3,000 g at 4ºC. Washing was repeated by adding 6 mL of ice-cold PEB, followed by centrifugation. Cells were then resuspended in 700 uL of ice-cold PEB, and transferred into pre-chilled 2 mL Eppendorf tubes containing 450 uL of pre-chilled RNAse-free 425-600 μm diameter glass beads (Sigma G8772). Cells were lysed by vortexing for 5 min at 4 ºC, followed by centrifugation at maximum speed at bench centrifuge for 5 min at 4ºC. 10% of the supernatant was aliquoted into Trizol for total RNA isolation, and kept at -80ºC. The remaining volume, corresponding approximately to 8 x 10e8 cells, was subsequently loaded onto the sucrose gradient. Linear sucrose gradients of 10-50% were prepared using the Gradient Station (BioComp). Briefly, SW41 centrifugation tubes (Beckman, Ultra-ClearTM) were filled with Gradient Solution 1 (GS1), which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 10% w/v RNAse-free sucrose. The tube was then filled with 6.3 mL of Gradient Solution 2 (GS2) layered at the bottom of the tube, which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 50% w/v RNAse-free sucrose. The linear gradient was formed using the tilted methodology, with the Gradient Station Maker (Biocomp). Once the gradients were formed, 400 µL of each lysate was carefully loaded on top of the gradients, and tubes were balanced in pairs, placed into pre-chilled SW41 buckets and centrifuged for 150 min, at 35,000 RPM. Gradients were then immediately fractionated using the Gradient Station, and 20 x 500 uL fractions were collected in 1.5 mL Eppendorf tubes, while absorbance was monitored at 260 nm continuously. Fractions were combined in the following way: the free rRNA (F1), the unassembled subunits (F2), the lowly-translating monosomes (F3) and the highly-translating polysomes (F4). The pooled fractions were then concentrated using Amicon-Ultra 100K columns (Millipore), and washed two times with PEB. The final volume was brought down to 200 µL, and RNA was extracted using TRIzol reagent.
Yeast total RNA was first treated with T4 Polynucleotide Kinase (PNK) (NEB M0201S)  in order to remove possible phosphorylated 3’ends before polyA tailing. Briefly, we mixed 1 ul PNK enzyme with rRNAs in a 50 ul reaction with T4 PNK Buffer (10X) and incubated at 37°C for 30 minutes. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. Dephosphorylated rRNAs were then polyadenylated using E.coli Poly(A) Polymerase, following the commercial protocol. Four different direct RNA libraries were barcoded according to the recent protocol that our lab has recently published [58]. Custom RT adaptors (IDT) were annealed using following conditions: custom Oligo A and B (Table S4) were mixed in annealing buffer (0.01M Tris-Cl pH7.5, 0.05M NaCl) to the final concentration of 1.4 uM each in a total volume of 75 ul. The mixture was incubated at 94 °C for 5 minutes and slowly cooled down (-0.1 °C/s) to room temperature. RNA library for direct RNA Sequencing (SQK-RNA002) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9080_v2_revI_14Aug2019 with half reaction for each library until the RNA Adapter (RMX) ligation step. Per reaction (half), 250 ng total of polyA tailed yeast rRNAs were ligated to pre-annealed custom RT adaptors (IDT) [58] (Table S4) using concentrated T4 DNA Ligase (NEB-M0202T), and was reverse transcribed using Maxima H Minus RT (Thermo Scientific, EP0752), without heat inactivation step. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. 50 ng of reverse transcribed RNA from each reaction was pooled and RMX adapter, composed of sequencing adapters with motor protein, was ligated onto the RNA:DNA hybrid and the mix was purified using 1X Agencourt RNAClean XP beads, washing with Wash Buffer (WSB) twice. The sample was then eluted in Elution Buffer (EB) and mixed with RNA Running Buffer  (RRB) prior to loading onto a primed R9.4.1 flow cell, and ran on a MinION sequencer with MinKNOW acquisition software version v.3.5.5..
Nanopore Direct RNA Sequencing

value: WT

tag: sucrose gradient fraction

value: Fraction 3-4

tag: rna fraction

value: Ribosomal RNA

tag: stress

value: H2O2 Stress


title: Yeast_rRNA_H202translated.rep1

description: Sample 20

tag: strain


title: 24h saha rep1

description:
replicate 1
22_24h_SAHA_R1_5617835006

tag: treatment


title: Yeast_rRNA_WTtranslated.rep2

description: Sample 23

tag: genotype

value: Normal


title: 24h dmso rep2

description:
replicate 2
29_24h_DMSO_R2_5617835004


title: Yeast_rRNA_H202input.rep2

description: Sample 22

value: Total RNA


title: Yeast_rRNA_WTinput.rep2

description: Sample 21


title: 00078A0041_PL


title: 00078A0058_PL


title: Lenti_Control_2_Day7_B10


title: Yeast_rRNA_WTtranslated.rep1

description: Sample 19


title: Yeast_rRNA_H202input.rep1

description: Sample 18


title: 00078A002C_PL


title: Lenti_Control_2_Day7_B11


title: Yeast_rRNA_PolysomeF4.rep2

description: Sample 16

value: Fraction 4


title: Yeast_rRNA_WTinput.rep1

description: Sample 17


title: Yeast_rRNA_PolysomeF3.rep2

description: Sample 15

value: Fraction 3


title: Yeast_rRNA_PolysomeF2.rep2

description: Sample 14

value: Fraction 2


title: 24h dmso rep1

description:
replicate 1
28_24h_DMSO_R1_5617835004


title: Lenti_Control_2_Day7_B09


title: Lenti_Control_2_Day7_B08


title: Yeast_rRNA_PolysomeF4.rep1

description: Sample 12


title: 000789FFF0_PL

value: Heterogeneous Stock

value: M


title: Yeast_rRNA_PolysomeF1.rep2

description: Sample 13

value: Fraction 1


title: 000789FFF8_PL

value: Prelimbic cortex


title: 000789FFD3_PL


title: Yeast_rRNA_PolysomeF3.rep1

description: Sample 11


title: Yeast_rRNA_PolysomeF2.rep1

description: Sample 10


title: Lenti_Control_2_Day7_B07


title: Yeast_rRNA_PolysomeF1.rep1

description: Sample 9


title: 000789FFAC_PL

description: Removed: expression outlier


title: Lenti_Control_2_Day7_B06


title: Lenti_Control_2_Day7_B05


title: Yeast_rRNA_snR34KO.rep2

description: Sample 6

extract_protocol:
For the samples 1-8 and 17-18-21-22:Saccharomyces cerevisiae BY4741 cells (strains: snR3Δ, snR34Δ snR36Δ, and WT) were harvested via centrifugation at 3000 RPM for 1 minute, followed by two washes with water. RNA was purified from pelleted cells using a MasterPure Yeast RNA extraction kit (Lucigen, MPY03100), according to manufacturer’s instructions., For the rest (Fractions): Yeast pellets from 100 mL cultures were washed with 6 mL of ice-cold Polysome Extraction Buffer (PEB), which contained 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 100 U/mL RNAse inhibitors (RNaseOUT, Invitrogen, #18080051). Cells were centrifuged for 5 min at 3,000 g at 4ºC. Washing was repeated by adding 6 mL of ice-cold PEB, followed by centrifugation. Cells were then resuspended in 700 uL of ice-cold PEB, and transferred into pre-chilled 2 mL Eppendorf tubes containing 450 uL of pre-chilled RNAse-free 425-600 μm diameter glass beads (Sigma G8772). Cells were lysed by vortexing for 5 min at 4 ºC, followed by centrifugation at maximum speed at bench centrifuge for 5 min at 4ºC. 10% of the supernatant was aliquoted into Trizol for total RNA isolation, and kept at -80ºC. The remaining volume, corresponding approximately to 8 x 10e8 cells, was subsequently loaded onto the sucrose gradient. Linear sucrose gradients of 10-50% were prepared using the Gradient Station (BioComp). Briefly, SW41 centrifugation tubes (Beckman, Ultra-ClearTM) were filled with Gradient Solution 1 (GS1), which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 10% w/v RNAse-free sucrose. The tube was then filled with 6.3 mL of Gradient Solution 2 (GS2) layered at the bottom of the tube, which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 50% w/v RNAse-free sucrose. The linear gradient was formed using the tilted methodology, with the Gradient Station Maker (Biocomp). Once the gradients were formed, 400 µL of each lysate was carefully loaded on top of the gradients, and tubes were balanced in pairs, placed into pre-chilled SW41 buckets and centrifuged for 150 min, at 35,000 RPM. Gradients were then immediately fractionated using the Gradient Station, and 20 x 500 uL fractions were collected in 1.5 mL Eppendorf tubes, while absorbance was monitored at 260 nm continuously. Fractions were combined in the following way: the free rRNA (F1), the unassembled subunits (F2), the lowly-translating monosomes (F3) and the highly-translating polysomes (F4). The pooled fractions were then concentrated using Amicon-Ultra 100K columns (Millipore), and washed two times with PEB. The final volume was brought down to 200 µL, and RNA was extracted using TRIzol reagent.
Yeast total RNA was first treated with T4 Polynucleotide Kinase (PNK) (NEB M0201S)  in order to remove possible phosphorylated 3’ends before polyA tailing. Briefly, we mixed 1 ul PNK enzyme with rRNAs in a 50 ul reaction with T4 PNK Buffer (10X) and incubated at 37°C for 30 minutes. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. Dephosphorylated rRNAs were then polyadenylated using E.coli Poly(A) Polymerase, following the commercial protocol. Four different direct RNA libraries were barcoded according to the recent protocol that our lab has recently published [58]. Custom RT adaptors (IDT) were annealed using following conditions: custom Oligo A and B (Table S4) were mixed in annealing buffer (0.01M Tris-Cl pH7.5, 0.05M NaCl) to the final concentration of 1.4 uM each in a total volume of 75 ul. The mixture was incubated at 94 °C for 5 minutes and slowly cooled down (-0.1 °C/s) to room temperature. RNA library for direct RNA Sequencing (SQK-RNA002) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9080_v2_revI_14Aug2019 with half reaction for each library until the RNA Adapter (RMX) ligation step. Per reaction (half), 250 ng total of polyA tailed yeast rRNAs were ligated to pre-annealed custom RT adaptors (IDT) [58] (Table S4) using concentrated T4 DNA Ligase (NEB-M0202T), and was reverse transcribed using Maxima H Minus RT (Thermo Scientific, EP0752), without heat inactivation step. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. 50 ng of reverse transcribed RNA from each reaction was pooled and RMX adapter, composed of sequencing adapters with motor protein, was ligated onto the RNA:DNA hybrid and the mix was purified using 1X Agencourt RNAClean XP beads, washing with Wash Buffer (WSB) twice. The sample was then eluted in Elution Buffer (EB) and mixed with RNA Running Buffer  (RRB) prior to loading onto a primed R9.4.1 flow cell, and ran on a MinION sequencer with MinKNOW acquisition software version v.3.5.5..
Nanopore Direct RNA Sequencing
Flongle adapter was used.


title: 000789FF94_PL


title: Yeast_rRNA_snR36KO.rep2

description: Sample 7


title: Yeast_rRNA_snR3KO.rep2

description: Sample 5


title: Yeast_rRNA_WT.rep2

description: Sample 8


title: Lenti_Control_2_Day7_B04


title: 000789FF7D_PL


title: Lenti_Control_2_Day7_B03


title: Yeast_rRNA_snR36KO.rep1

description: Sample 3


title: Yeast_rRNA_WT.rep1

description: Sample 4


title: 000789FFF9_PL


title: Yeast_rRNA_snR34KO.rep1

description: Sample 2


title: 000789FF6E_PL


title: 00077EA7E6_PL


title: Lenti_Control_2_Day7_B02


title: 00077E8336_PL


title: Lenti_Control_2_Day7_B01


title: 00077E76FE_PL


title: Lenti_Control_2_Day7_A10


title: Lenti_Control_2_Day7_A11


title: 00077E67B5_PL


title: 00078A261F_LHB

source_name: Brain region: Lateral habenula

value: Lateral habenula


title: 00078A2595_LHB


title: Lenti_Control_2_Day7_A08


title: Lenti_Control_2_Day7_A09


title: 00078A2496_LHB


title: Lenti_Control_2_Day7_A07


title: 00078A2463_LHB


title: 00078A2315_LHB


title: Lenti_Control_2_Day7_A05


title: Lenti_Control_2_Day7_A04


title: 00078A22EB_LHB


title: 00078A22DF_LHB


title: 00078A1F34_LHB


title: Lenti_Control_2_Day7_A02


title: Lenti_Control_2_Day7_A03


title: Yeast_rRNA_snR3KO.rep1

description: Sample 1


title: Lenti_Control_2_Day7_A12


title: Lenti_Control_2_Day7_A01


title: 00077E76C6_PL

description: Removed: expression outlier


title: 00078A1A2B_LHB


title: 00078A1B05_LHB


title: 00078A1A16_LHB


title: Lenti_Control_1_Day7_H11


title: Lenti_Control_2_Day7_A06


title: 00078A00BF_PL


title: Lenti_Control_1_Day7_H09


title: Lenti_Control_1_Day7_H08


title: 00078A19D6_LHB


title: Lenti_Control_1_Day7_H10


title: 00078A19C0_LHB


title: 00078A19B7_LHB

description: Removed: low read count


title: 00078A19A7_LHB


title: Lenti_Control_1_Day7_H07


title: Lenti_Control_1_Day7_H06


title: 00078A194B_LHB


title: Lenti_Control_1_Day7_H04


title: Lenti_Control_1_Day7_H05


title: 00078A19B5_LHB


title: 00078A1979_LHB


title: Lenti_Control_1_Day7_H12


title: 00078A193E_LHB


title: 00078A1937_LHB


title: Domestic chicken splenic-derived lymphocytes (Sample4077)

city: Houston

first: Olga

last: Dudchenko

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

data_processing:
Hi-C-guided genome assembly (3D-DNA, JBAT)
Construction of the chromosome-length Hi-C contact map (3D-DNA, Juicer)
Centromere position identification (repeat sequence mapping or structural analysis, 3D-DNA/JBAT)
Aggregate chromosome analysis (3D-DNA)
Variant calling (DRAGEN pipeline)
Chromosome-length variant phasing (3D-DNA)
Genome_build: In addition to those generate as part of the study, the following existing genome builds were used: GRCg6a (domestic chicken); Xla.v91 (African clawed frog); AaegL5.0 (Yellow fever mosquito), CpipJ3 (Southern domestic mosquito); Release_6_plus_ISO1_MT (fruit fly, with chromosome arms merged for ACA analysis resulting in Release_6_plus_ISO1_MT_merged); WBcel235 (C. elegans); sacCer3 (baker's yeast); arahy.Tifrunner.gnm2.J5K5 (ground nut); 161010_Chinese_Spring_v1.0_pseudomolecules (bread wheat).
Supplementary_files_format_and_content: fasta.gz: chromosome-length nucleotide sequences generated usign Hi-C-guided genome assembly
Supplementary_files_format_and_content: hic: contact maps generated by aligning Hi-C data to corresponding chromosome-length reference sequences. In addition to standard contact maps generated for all genomic positions across chromosomes the uploaded files include ACA (aggregate chromosome analysis) maps and phasing contact maps representing interactions between positions on the genome that contain heterogyzous SNPs. For more details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: wig: tracks showing the alignment frequency of Hi-C reads from read pairs that contain centromere repeat-specifc sequences.
Supplementary_files_format_and_content: bedpe: Files describing the tentative position of centromeres in chromosome-length genome assemblies, identified either by mapping repeat sequences associated with the centromere or via analysis of the contact map for evidence of centromere-to-telomere folding axis. For details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: vcf.gz: Variants (specifically SNPs) identivied by the DRAGEN variant calling pipeline from aligning Hi-C data to chromosome-length references generated via Hi-C-guided assembly and phased using the 3D-DNA Hi-C-guided phasing module.

organism: Gallus gallus

source_name: splenic-derived lymphocytes

extract_protocol:
Extraction protocol is described as part of the library construction protocol.
The Hi-C libraries were prepared following the in situ Hi-C strategy (Rao, Huntley et al., 2014). Briefly, cells or tissue were crosslinked and then lysed with nuclei permeabilized but still intact. DNA was then restricted and the overhangs filled in incorporating a biotinylated base. Free ends were then ligated together in situ. Crosslinks were reversed, the DNA was sheared to 300-500bp and then biotinylated ligation junctions were recovered with streptavidin beads. Afterward DNA was handled according to standard Illumina library construction protocol. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3' to 5' exo-) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3 end. After adapter ligation DNA was PCR amplified with Illumina primers and library fragments of 400-600 bp (insert plus adaptor and PCR primer sequences) were purified using SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on various Illumina instruments following the manufacturer's protocols. Fpr the tammar wallaby, a short insert-size PCR-free DNA-Seq library was prepared following the ClaSeek protocol.

tag: tissue

value: splenic-derived lymphocytes


title: Lenti_Control_1_Day7_H01


title: Lenti_Control_1_Day7_H03


title: Lenti_Control_1_Day7_H02


title: Lenti_Control_1_Day7_G12


title: Lenti_Control_1_Day7_G11


title: 00078A1905_LHB

tag: Sex


title: Lenti_Control_1_Day7_G10


title: Lenti_Control_2_Day7_C03


title: 00078A18F7_LHB


title: 00078A18CF_LHB

value: F


title: 00078A18A7_LHB

description: Removed: genotype mismatch


title: 00078A1942_LHB


title: 00078A189F_LHB


title: 00078A1875_LHB


title: Lenti_Control_1_Day7_G08


title: Lenti_Control_1_Day7_G07


title: Lenti_Control_1_Day7_G05


title: Lenti_Control_1_Day7_G04


title: Lenti_Control_1_Day7_G03


title: Lenti_Control_1_Day7_G02


title: 00078A1837_LHB


title: 00078A186C_LHB


title: Spleen, poor swimmer, respiration, fish 10

city: Aas

first: Aleksei

last: Krasnov

address: Osloveien 1

department: Fish Health

country: Norway

institute: Nofima AS

hyb_protocol: Hybridized according to Agilent Two-Color Microarray-Based Gene Expression Analysis protocol.

scan_protocol: Arrays were scanned with GenePix 4100 microarray scanner.

data_processing: Images were processed using GenePix Pro 6.0. After removal of flagged spots, lowess normalization was performed.

organism: Salmo salar

source_name: Spleen

label_protocol: Amplified and labeled with Low Input Quick Amp Labeling Kit (Agilent).

growth_protocol: Salmon was fed with commercial feed.

extract_protocol: Total RNA was extracted using the RNeasy® Mini Kit (Qiagen).

value: Spleen

tag: swimming performance

value: Poor

tag: condition

value: Respiration

source_name: Spleen, equalized mixture

tag: sample type

value: Reference


title: 00078A1863_LHB


title: 00078A192C_LHB


title: Spleen, poor swimmer, respiration, fish 6


title: Spleen, poor swimmer, respiration, fish 5


title: 00078A181B_LHB


title: Spleen, poor swimmer, respiration, fish 9


title: Spleen, poor swimmer, respiration, fish 8


title: Lenti_Control_1_Day7_G06


title: Lenti_Control_1_Day7_G01


title: 00078A1816_LHB

description: Removed: low read count


title: Spleen, poor swimmer, respiration, fish 1


title: Spleen, poor swimmer, respiration, fish 2


title: Spleen, poor swimmer, control, fish 4

value: Control


title: Spleen, poor swimmer, respiration, fish 7


title: Spleen, poor swimmer, respiration, fish 4


title: Lenti_Control_1_Day7_F11


title: Spleen, poor swimmer, control, fish 3


title: 00078A17F7_LHB


title: 00078A1807_LHB


title: Lenti_Control_1_Day7_F10


title: Spleen, poor swimmer, respiration, fish 3


title: Lenti_Control_1_Day7_G09


title: Lenti_Control_1_Day7_F12


title: Spleen, poor swimmer, control, fish 2


title: 00078A1772_LHB


title: Lenti_Control_1_Day7_F09


title: 00078A179C_LHB


title: Lenti_Control_1_Day7_F08


title: Lenti_Control_1_Day7_F07


title: Lenti_Control_1_Day7_F06


title: Spleen, poor swimmer, control, fish 1


title: 00078A1731_LHB


title: 00078A1732_LHB


title: Lenti_Control_1_Day7_F05


title: Spleen, poor swimmer, E_Crit, fish 6


title: 00078A1707_LHB


title: 00078A16DF_LHB


title: Lenti_Control_1_Day7_F03


title: Spleen, poor swimmer, E_Crit, fish 10


title: 00078A16D3_LHB


title: Lenti_Control_1_Day7_F02


title: 00078A0AEA_LHB


title: Spleen, poor swimmer, E_Crit, fish 7


title: Lenti_Control_1_Day7_F01


title: 00078A09B1_LHB


title: Lenti_Control_1_Day7_F04


title: Spleen, poor swimmer, E_Crit, fish 4


title: Spleen, poor swimmer, E_Crit, fish 5


title: Lenti_Control_1_Day7_E11


title: 00078A096C_LHB


title: Spleen, poor swimmer, E_Crit, fish 8


title: Spleen, poor swimmer, E_Crit, fish 3


title: Lenti_Control_1_Day7_E10


title: 00078A0A43_LHB


title: 00078A087B_LHB


title: Spleen, poor swimmer, E_Crit, fish 1


title: Lenti_Control_1_Day7_E12


title: Spleen, poor swimmer, E_Crit, fish 9


title: Spleen, poor swimmer, E_Crit, fish 2


title: 00078A07A2_LHB


title: Lenti_Control_1_Day7_E08


title: 00078A02DF_LHB


title: 00078A02CB_LHB


title: Lenti_Control_1_Day7_E07


title: Lenti_Control_1_Day7_E09


title: Lenti_Control_1_Day7_E06


title: 00078A0255_LHB


title: Lenti_Control_1_Day7_E05


title: 00078A0246_LHB

description: Removed: expression outlier


title: Lenti_Control_1_Day7_E04


title: Spleen, good swimmer, respiration, fish 9

value: Good


title: Spleen, good swimmer, respiration, fish 10


title: Spleen, good swimmer, respiration, fish 7


title: Spleen, good swimmer, respiration, fish 8


title: Spleen, good swimmer, respiration, fish 4


title: Spleen, good swimmer, respiration, fish 5


title: Spleen, good swimmer, respiration, fish 2


title: Spleen, good swimmer, respiration, fish 6


title: 00078A0244_LHB

description: Removed: low read count


title: Spleen, good swimmer, respiration, fish 3


title: Lenti_Control_1_Day7_E03


title: Lenti_Control_1_Day7_E02


title: 00078A022D_LHB


title: Lenti_Control_1_Day7_D12


title: Spleen, good swimmer, respiration, fish 1


title: 00078A0224_LHB


title: 00078A021A_LHB


title: Spleen, good swimmer, control, fish 4


title: 00078A0215_LHB


title: Lenti_Control_1_Day7_D11


title: Spleen, good swimmer, control, fish 3


title: Spleen, good swimmer, control, fish 2


title: Spleen, good swimmer, control, fish 1


title: 00078A17A7_LHB


title: Lenti_Control_1_Day7_D10


title: AC066_Gingivitis (array)

type: genomic

hyb_protocol: Bisulfite-conversion was carried out using the Epitect 96 Bisulfite Kit (Qiagen, Hilden, Germany), and 300ng of converted DNA were hybridized to Illumina Infinium MethylationEPIC BeadChips, following the the manufacturer’s instructions.

scan_protocol: Arrays were imaged using iScan system following the standard Illumina instructions.

data_processing: The methylation data were exported from Illumina GenomeStudio as IDAT files. Then the samples (idat files) were normalized and preprocessed using minfi (v1.24.0) package of R (v3.4.2).

label: Cy5 and Cy3

label_protocol: The tissue samples were stored in labeled individual Eppendorf tubes containing Allprotect Tissue Reagent (Qiagen, USA). Total DNA were isolated using an AllPrep DNA Kit (Qiagen, USA).

extract_protocol: gingival tissue samples were harvested in conjunction with an invasive oral surgical procedure required for the participant’s oral care after administration of local anesthesia.

value: Gingival tissue from gingivitis

tag: age

tag: race/ethnicity

value: White

tag: batch

value: B

tag: phenotype

value: Gingivitis


title: Spleen, good swimmer, E_Crit, fish 10


title: 00078A01DB_LHB

tag: population

value: Heterogeneous Stock

tag: rat batch

tag: brain region


title: AC072_Gingivitis (array)

value: Arfican-American


title: 00078A01D8_LHB

value: M


title: AC064_Gingivitis (array)


title: Spleen, good swimmer, E_Crit, fish 9


title: Lenti_Control_1_Day7_D08


title: 00078A01C0_LHB


title: Lenti_Control_1_Day7_E01


title: AC068_Gingivitis (array)


title: Lenti_Control_1_Day7_D07


title: AC054_Healthy (array)

value: Healthy gingival tissue

value: Asian

value: Healthy


title: 00078A01A6_LHB

value: Lateral habenula


title: 00078A01FE_LHB

value: F


title: AC070_Gingivitis (array)

value: Hispanic/Latino


title: Spleen, good swimmer, E_Crit, fish 7

value: Spleen


title: Lenti_Control_1_Day7_D06


title: AC062_Gingivitis (array)


title: AC060_Healthy (array)


title: AC058_Healthy (array)


title: AC056_Healthy (array)


title: Lenti_Control_1_Day7_D05


title: Spleen, good swimmer, E_Crit, fish 6

value: Reference


title: Spleen, good swimmer, E_Crit, fish 5


title: 00078A0166_LHB


title: Lenti_Control_1_Day7_D04


title: 00078A0139_LHB


title: 00078A0138_LHB


title: AC048_Periodontitis (array)

value: Gingival tissue from periodontitis

value: Periodontitis


title: AC046_Periodontitis (array)


title: Lenti_Control_1_Day7_D02


title: Spleen, good swimmer, E_Crit, fish 3

value: Good


title: Spleen, good swimmer, E_Crit, fish 8


title: Spleen, good swimmer, E_Crit, fish 4


title: Lenti_Control_1_Day7_D09


title: AC052_Healthy (array)

value: White


title: 00078A0189_LHB


title: AC044_Periodontitis (array)

value: B


title: Lenti_Control_1_Day7_D01


title: AC050_Healthy (array)


title: Lenti_Control_1_Day7_D03


title: Lenti_Control_1_Day7_C11


title: Lenti_Control_1_Day7_C12


title: 00078A0127_LHB


title: Lenti_Control_1_Day7_C10


title: 00078A0085_LHB


title: Heart, poor swimmer, respiration, fish 9

source_name: Heart

value: Heart

value: Poor

value: Respiration

source_name: Heart, equalized mixture


title: Heart, poor swimmer, respiration, fish 10


title: Heart, poor swimmer, respiration, fish 8


title: 00078A0041_LHB

value: Heterogeneous Stock


title: AC037_Periodontitis (array)


title: 00078A0058_LHB

value: M


title: AC034_Gingivitis (array)

value: Gingival tissue from gingivitis

value: A

value: Gingivitis


title: AC030_Gingivitis (array)

value: F


title: AC032_Gingivitis (array)

value: Asian


title: Spleen, good swimmer, E_Crit, fish 2

value: Spleen


title: AC042_Periodontitis (array)


title: 00078A002C_LHB

value: Lateral habenula


title: AC039_Periodontitis (array)

value: Gingival tissue from periodontitis


title: 00078A00BF_LHB


title: Heart, poor swimmer, respiration, fish 7

value: Reference


title: Spleen, good swimmer, E_Crit, fish 1

value: Good


title: 00078A00AC_LHB


title: AC026_Gingivitis (array)

value: White


title: Lenti_Control_1_Day7_C07


title: Lenti_Control_1_Day7_C09


title: AC024_Healthy (array)

value: Healthy gingival tissue

value: Healthy


title: 000789FFF9_LHB

description: Original label before genotype mismatch fix: 000789FFF0_LHB


title: Lenti_Control_1_Day7_C06


title: AC028_Gingivitis (array)


title: 000789FFF8_LHB


title: Heart, poor swimmer, respiration, fish 5

value: Heart

value: Poor


title: Roundworm whole animal(s) (Sample4082)

organism: Caenorhabditis elegans

source_name: whole animals

value: whole animals


title: Lenti_Control_1_Day7_C05


title: 000789FFF0_LHB

description: Original label before genotype mismatch fix: 000789FFF9_LHB

value: Heterogeneous Stock

value: M


title: Heart, poor swimmer, respiration, fish 6

value: Respiration


title: Heart, poor swimmer, respiration, fish 3


title: Lenti_Control_1_Day7_C04


title: PDS-treated cells - igG_rep1

city: Moscow

first: Artem

last: Luzhin

address: Vavilova 34/5

country: Russia

institute: Institute of Gene Biology RAS

data_processing:
ChIP-seq reads were aligned to the using Bowtie2 (version 2.2.3) with the ‘—very-sensitive’ preset.
Non-uniquely mapped reads, PCR duplicates and reads with MAPQ < 30 were filtered out with 'samtools rmdup' and 'samtools view -h -F 256 -q 30'
Peaks were called using PePr (https://github.com/shawnzhangyx/PePr) with a p-value cutoff of 0.05 and a sliding window size of 100 bp
The bigWig files were generated using deepTools, version 2.0
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files; Scores represent ChIP to IgG ratio
Supplementary_files_format_and_content: CTCF peaks bed files

source_name: Immortalised myelogenous leukemia

growth_protocol: K562 cells were cultured in RMPI-1640 medium (Paneco, Russia) supplemented with 0.4%  fetal bovine serum (HyClone GE Healthcare, USA).

extract_protocol: The immunoprecipitated samples and the inputs were subjected to Nextera library preparation as described by the manufacturer (NEBNext Ultra II DNA library Prep Kit for Illumina, New England Biolabs). The quality and size of libraries and chromatin shearing fragments were checked by Agilent Bioanalyzer using Agilent DNA High Sensitivity Chips (Agilent Technologies). Samples were sequenced on an Illumina HiSeq 1500 platform in single-end using 50-bp reads. The experiment was repeated twice.

treatment_protocol: The G4-stablizing ligands (PDS and PhenDC3) were added to the cell suspensions (1-1.25 mln/mL) to final concentrations of 10 or 20 μM (PDS) and 8 μM (Phen DC3). After 24 h (10 μM PDS) or 48 h (20 μM PDS or 8 μM PhenDC3) of incubation, cells were fixed, and chromatin immunoprecipitation (ChIP) was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit with magnetic beads (Cell Signalling Technology, USA) following the manufacturer’s protocol.

tag: cell line

value: K562 cells

value: treated with PDS

tag: antibody

value: IgG

tag: cell type

value: erythroleukemia


title: 000789FFD3_LHB

value: Lateral habenula


title: Lenti_Control_1_Day7_C03

source_name: Glioblastoma single cell

tag: strain


title: PDS-treated cells - igG_rep2

tag: treatment


title: Heart, poor swimmer, respiration, fish 2

tag: tissue


title: Lenti_Control_1_Day7_C08


title: Heart, poor swimmer, respiration, fish 1


title: Lenti_Control_1_Day7_C02


title: PDS-treated cells - CTCF_rep2

value: CTCF


title: AC016_Healthy (array)

tag: Sex

value: F

value: Arfican-American

value: A


title: PDS-treated cells - CTCF_rep1


title: AC035_Gingivitis (array)

value: Gingival tissue from gingivitis

value: Hispanic/Latino

value: Gingivitis


title: 000789FF94_LHB


title: Lenti_Control_1_Day7_C01


title: 000789FFAC_LHB


title: Non-treated cells - IgG_rep1

value: untreated


title: Heart, poor swimmer, control, fish 4

value: Heart

tag: swimming performance

value: Poor

value: Control

value: Reference


title: 000789FF6E_LHB

value: Heterogeneous Stock


title: Heart, poor swimmer, control, fish 3

tag: condition


title: AC018_Healthy (array)

value: Healthy gingival tissue

value: Healthy


title: AC019_Healthy (array)

value: M

value: White


title: Heart, poor swimmer, control, fish 2

tag: sample type


title: AC022_Healthy (array)

tag: age


title: 000789FF7D_LHB

value: Lateral habenula


title: Lenti_Control_1_Day7_B10


title: Lenti_Control_1_Day7_B12


title: Lenti_Control_1_Day7_B11


title: Heart, poor swimmer, control, fish 1


title: Non-treated cells - CTCF_rep1

value: K562 cells

value: CTCF

value: erythroleukemia


title: 00077EA7E6_LHB


title: 00077E8336_LHB


title: P4P6 Chemical Mapping - NM

data_processing:
Library strategy: Barcode amplicon sequencing
Demultiplexed reads were downloaded from the BaseSpace Sequence Hub (Illumina). Most analysis steps were carried out using the RNAFramework RNA structure probing analysis toolkit. Using the `rf-map` module, reads were trimmed with CutAdapt and mapped to the wild-type RNA sequence using Bowtie 2. Mutations were counted using the `rf-count` module with `-m` flag, then normalized using the `rf-norm` module with `-sm 4 -nm 2 -rb AC -nw 50 -dw` flags for DMS samples and `-sm 3 -nm 2 -rb N -nw 50 -dw` flags for 1M7 samples. These normalized reactivities were plotted as-is, and also used to predict RNA secondary structure using the RNAStructure `Fold` command, implemented in Arnie (https://github.com/DasLab/arnie).
processed data files format and content: Table S4 contains normalized nucleotide-resolution reactivities of YellowStone, LinearDesign, and P4P6 (positive control) constructs. DMS reactivity signals are provided for all three constructs, and 1M7 reactivity signals are provided for YellowStone and LinearDesign.

extract_protocol:
In vitro treated RNAs were purified by Zymo RNA Clean and Concentrator 5 kit.
10 µL of the modified RNA was mixed with 1 µL of 10 µM oVT555 (GACGCCGTACGTACGCGG), incubated at 65 °C for 5 min, and snap cooled on ice. Then, the template-primer mix was combined with 4 µL of 5X TGIRT First Strand Synthesis Buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2 ), 2 µL 10 mM dNTPs, 1 µL freshly prepared 100 mM DTT, and 0.5 µL TGIRT-III (InGex LLC). The reaction was mixed and incubated at 57 °C for 3 hours. RNA was then hydrolyzed by addition of 10 µL hydrolysis buffer (0.5 M NaOH, 0.25 M EDTA) and incubation at 65 °C for 15 min. Hydrolysis was quenched by bringing the reaction volume to 50 µL with water, adding 100 µL of Oligo Binding Buffer (Zymo Research), and proceeding through the Zymo Oligo Clean and Concentrator (Zymo Research) purification protocol, eluting in 15 µL of water. 5 µL of the purified cDNA was amplified in a NEBNext Q5 HotStart master mix PCR reaction containing 0.5 µM each oVT554 (GGGACATTTGCTTCTGACACAACTGTGTTCA) and oVT555 (GACGCCGTACGTACGCGG) with the following cycling conditions: 98 °C for 30 seconds, 10 cycles of 98 °C for 10 seconds followed by 72 °C for 60 seconds, with a final extension of 72 °C for 5 minutes. Products were purified using 0.9X Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research).Amplification of the full-length cDNA product was verified on an agarose gel stained with SYBRSafe (Invitrogen). Double-stranded cDNA was prepared for Illumina sequencing with the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) and iTru primers. Briefly, 100-500ng of DNA from either 1M7 or DMS conditions in 26 µL of water was fragmented and end-repaired by adding 7 µL NEBNext Ultra II FS Reaction Buffer and 2 µL NEBNext Ultra II FS Enzyme Mix, mixed thoroughly by vortexing, and incubated in a thermocycler with heated lid on at 37°C for 20 min, then 65°C for 20 min. Fresh ligation adapter was prepared by denaturing a solution of 15 µM each of iTrusR1-stub (ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) and iTrusR2-stubRCp (/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCA*C) in salty TLE (10 mM Tris pH 8.0, 0.1 mM EDTA, 100 mM NaCl) at 95°C for 1 minute, then annealed via slow cooling at -0.1°C/s to 25°C. The fragmented and end-repaired DNA was mixed with 2.5 µL 15 µM ligation adapter, 30 µL NEBNext Ultra II FS Ligation Master Mix, and 1 µL NEBNext Ultra II FS Ligation Enhancer. The ligation reaction was incubated in a thermocycler (heated lid off) at 20°C for 60 min. Ligated fragments were purified with 0.9X Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research) and eluted in 25 µL water. Dual-index sample barcodes were added through indexing PCR using the NEBNext Q5 Hot Start HiFi PCR Master Mix in a reaction with 0.5 µM each of iTru_5 and iTru_7 indexing primers with the following thermocycling parameters: 98°C for 30 s, 8 cycles of 98°C for 10 s, 55°C for 10 s, 72°C for 15 s, and a final extension at 72°C for 5 min. The final sequencing libraries were purified with 0.9X volumes of Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research)

value: in vitro transcribed

value: NM


title: Heart, poor swimmer, E_Crit, fish 9


title: Lenti_Control_1_Day7_B09


title: Lenti_Control_1_Day7_B08


title: 00077E76FE_LHB

description: Removed: expression outlier

value: F


title: AC013_Healthy (array)

tag: race/ethnicity

value: Hispanic/Latino

value: A


title: AC009_Periodontitis (array)

value: Gingival tissue from periodontitis

value: Indian

tag: batch

value: Periodontitis


title: P4P6 Chemical Mapping - DMS

value: DMS


title: Heart, poor swimmer, respiration, fish 4

value: Heart

value: Poor

value: Respiration

value: Reference


title: AC007_Periodontitis (array)

tag: phenotype


title: Heart, poor swimmer, E_Crit, fish 8


title: 00077E76C6_LHB

tag: population

value: Heterogeneous Stock

value: Lateral habenula


title: C1 Chemical Mapping - DMS


title: Lenti_Control_1_Day7_B06


title: Lenti_Control_1_Day7_B07


title: AC011_Periodontitis (array)

value: White


title: Lenti_Control_1_Day7_B05


title: Heart, poor swimmer, E_Crit, fish 6


title: 00078A2667_IL

description: Removed: genotype mismatch

source_name: Brain region: Infralimbic cortex

value: M

tag: rat batch

value: Infralimbic cortex


title: Heart, poor swimmer, E_Crit, fish 5


title: 00078A261F_IL

tag: brain region


title: Heart, poor swimmer, E_Crit, fish 7


title: 00078A2595_IL


title: AC003_Periodontitis (array)


title: B1 Chemical Mapping - DMS

value: in vitro transcribed


title: AC005_Periodontitis (array)


title: Lenti_Control_1_Day7_B02


title: C1 Chemical Mapping - DMSO

extract_protocol:
In vitro treated RNAs were purified by Zymo RNA Clean and Concentrator 5 kit.
10 µL of modified RNA was mixed with 1 µL of 200 ng/µL random nonamer primer (New England Biolabs), incubated at 65°C for 5 minutes, then snap-cooled on ice. Then, 8 µL of 2.5X MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 25 mM DTT, 1.25 mM each dNTPs, 15 mM MnCl2), was added to the primer-template mixture, incubated at room temperature for 2 minutes, and then mixed with 1 µL of SuperScript II (Invitrogen). The reverse transcription reaction was thoroughly mixed by pipetting, incubated at room temperature for 10 minutes, then at 42 °C for 3 hours. The reverse transcription enzyme was heat inactivated at 70°C for 15 min, snap-cooled on ice, then immediately mixed with 8 µL NEBNext Second Strand Synthesis Reaction Buffer (New England Biolabs), 4 µL NEBNext Second Strand Synthesis Enzyme Mix (New England Biolabs), and 48 µL water. The second strand synthesis reaction was mixed thoroughly through pipetting and incubated in a thermocycler with the heated lid off at 16°C for 60 min. The resulting double-stranded cDNA was purified with 1.8X volumes of Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research). Double-stranded cDNA was prepared for Illumina sequencing with the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) and iTru primers. Briefly, 100-500ng of DNA from either 1M7 or DMS conditions in 26 µL of water was fragmented and end-repaired by adding 7 µL NEBNext Ultra II FS Reaction Buffer and 2 µL NEBNext Ultra II FS Enzyme Mix, mixed thoroughly by vortexing, and incubated in a thermocycler with heated lid on at 37°C for 20 min, then 65°C for 20 min. Fresh ligation adapter was prepared by denaturing a solution of 15 µM each of iTrusR1-stub (ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) and iTrusR2-stubRCp (/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCA*C) in salty TLE (10 mM Tris pH 8.0, 0.1 mM EDTA, 100 mM NaCl) at 95°C for 1 minute, then annealed via slow cooling at -0.1°C/s to 25°C. The fragmented and end-repaired DNA was mixed with 2.5 µL 15 µM ligation adapter, 30 µL NEBNext Ultra II FS Ligation Master Mix, and 1 µL NEBNext Ultra II FS Ligation Enhancer. The ligation reaction was incubated in a thermocycler (heated lid off) at 20°C for 60 min. Ligated fragments were purified with 0.9X Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research) and eluted in 25 µL water. Dual-index sample barcodes were added through indexing PCR using the NEBNext Q5 Hot Start HiFi PCR Master Mix in a reaction with 0.5 µM each of iTru_5 and iTru_7 indexing primers with the following thermocycling parameters: 98°C for 30 s, 8 cycles of 98°C for 10 s, 55°C for 10 s, 72°C for 15 s, and a final extension at 72°C for 5 min. The final sequencing libraries were purified with 0.9X volumes of Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research)

value: DMSO


title: Heart, poor swimmer, E_Crit, fish 4


title: 00077E67B5_LHB


title: C1 Chemical Mapping - 1M7


title: Lenti_Control_1_Day7_B01


title: Lenti_Control_1_Day7_B04


title: B1 Chemical Mapping - DMSO


title: Lenti_Control_1_Day7_B03


title: Heart, poor swimmer, E_Crit, fish 3


title: 00078A2315_IL

value: F


title: B1 Chemical Mapping - NM

value: NM


title: 00078A2496_IL

description: Removed: low read count


title: AC001_Periodontitis (array)

value: Gingival tissue from periodontitis

value: A


title: 00078A2463_IL


title: B1 Chemical Mapping - 1M7


title: Lenti_Control_1_Day7_A11


title: Lenti_Control_1_Day7_A10


title: 00078A22EB_IL


title: Heart, poor swimmer, E_Crit, fish 1


title: 00078A1F34_IL


title: Heart, poor swimmer, E_Crit, fish 10


title: Heart, poor swimmer, E_Crit, fish 2


title: 00078A1A2B_IL


title: 00078A1B05_IL


title: Heart, good swimmer, respiration, fish 9

value: Good


title: Heart, good swimmer, respiration, fish 8


title: Heart, good swimmer, respiration, fish 10


title: Lenti_Control_1_Day7_A07


title: C1 Chemical Mapping - NM


title: Lenti_Control_1_Day7_A06


title: 00078A1A16_IL


title: Lenti_Control_1_Day7_A05


title: Lenti_Control_1_Day7_A12


title: 00078A19C0_IL


title: Lenti_Control_1_Day7_A08


title: 00078A19D6_IL


title: Lenti_Control_1_Day7_A04


title: Lenti_Control_1_Day7_A09


title: Heart, good swimmer, respiration, fish 7


title: 00078A22DF_IL


title: Heart, good swimmer, respiration, fish 4


title: 00078A19B7_IL


title: Lenti_Control_1_Day7_A03


title: 00078A19B5_IL


title: Lenti_Control_1_Day7_A02


title: 00078A19A7_IL


title: Lenti_Control_1_Day7_A01


title: Heart, good swimmer, respiration, fish 1


title: Heart, good swimmer, respiration, fish 6


title: Heart, good swimmer, respiration, fish 2


title: Heart, good swimmer, respiration, fish 3


title: 00078A1979_IL


title: Heart, good swimmer, control, fish 4

value: Control


title: Heart, good swimmer, respiration, fish 5

value: Heart


title: 00078A1937_IL


title: Heart, good swimmer, E_Crit, fish 10


title: Non-treated cells - CTCF rep2

tag: cell line

value: K562 cells

value: untreated

value: CTCF

value: erythroleukemia


title: Heart, good swimmer, E_Crit, fish 9

value: Reference


title: Heart, good swimmer, control, fish 2


title: GBM8_batch_3283


title: GBM8_batch_3288


title: GBM8_batch_3287


title: 00078A1942_IL

value: Heterogeneous Stock


title: GBM8_batch_3282


title: GBM8_batch_3286


title: 00078A193E_IL

value: M


title: 00078A194B_IL

value: Infralimbic cortex


title: Heart, good swimmer, control, fish 1


title: GBM8_batch_3280


title: GBM8_batch_3285


title: GBM8_batch_3284


title: Heart, good swimmer, E_Crit, fish 6


title: 00078A18A7_IL

description: Removed: genotype mismatch


title: Heart, good swimmer, E_Crit, fish 7


title: GBM8_batch_3279


title: Heart, good swimmer, E_Crit, fish 8


title: 00078A1905_IL


title: 00078A1875_IL


title: Indian muntjac fibroblasts (Sample3923)

organism: Muntiacus muntjak

source_name: fibroblasts

value: fibroblasts


title: GBM8_batch_3278


title: 00078A18F7_IL


title: 00078A192C_IL


title: Heart, good swimmer, control, fish 3


title: 00078A1863_IL


title: Chinese muntjac fibroblasts (Sample2606)

organism: Muntiacus reevesi


title: GBM8_batch_3277


title: GBM8_batch_3281


title: 00078A18CF_IL


title: 00078A1807_IL


title: Heart, good swimmer, E_Crit, fish 5


title: Heart, good swimmer, E_Crit, fish 4


title: GBM8_batch_3271


title: 00078A179C_IL


title: 00078A189F_IL


title: 00078A186C_IL


title: GBM8_batch_3276


title: Heart, good swimmer, E_Crit, fish 3


title: GBM8_batch_3275


title: Indian muntjac (Sample2604, Sample3923)

description:
Sample2604, Sample3923
Some libraries sequenced with NextSeq 550


title: GBM8_batch_3274


title: 00078A1816_IL


title: 00078A1837_IL


title: GBM8_batch_3267


title: 00078A17A7_IL


title: GBM8_batch_3272


title: GBM8_batch_3266


title: GBM8_batch_3269


title: Heart, good swimmer, E_Crit, fish 1


title: 00078A181B_IL


title: Heart, good swimmer, E_Crit, fish 2


title: GBM8_batch_3265


title: Southern house mosquito whole animal(s) (SAMN06546149)

organism: Culex quinquefasciatus

source_name: whole adult mosquito

value: whole adult mosquito


title: 00078A1731_IL

value: F


title: GBM8_batch_3270


title: 00078A17F7_IL


title: 00078A1707_IL


title: 00078A1772_IL


title: GBM8_batch_3273


title: 00078A1732_IL


title: Tammar wallaby blood (Sample1096)

data_processing:
Library strategy: ClaSeek PCR-free DNA-Seq
Hi-C-guided genome assembly (3D-DNA, JBAT)
Construction of the chromosome-length Hi-C contact map (3D-DNA, Juicer)
Centromere position identification (repeat sequence mapping or structural analysis, 3D-DNA/JBAT)
Aggregate chromosome analysis (3D-DNA)
Variant calling (DRAGEN pipeline)
Chromosome-length variant phasing (3D-DNA)
Genome_build: In addition to those generate as part of the study, the following existing genome builds were used: GRCg6a (domestic chicken); Xla.v91 (African clawed frog); AaegL5.0 (Yellow fever mosquito), CpipJ3 (Southern domestic mosquito); Release_6_plus_ISO1_MT (fruit fly, with chromosome arms merged for ACA analysis resulting in Release_6_plus_ISO1_MT_merged); WBcel235 (C. elegans); sacCer3 (baker's yeast); arahy.Tifrunner.gnm2.J5K5 (ground nut); 161010_Chinese_Spring_v1.0_pseudomolecules (bread wheat).
Supplementary_files_format_and_content: fasta.gz: chromosome-length nucleotide sequences generated usign Hi-C-guided genome assembly
Supplementary_files_format_and_content: hic: contact maps generated by aligning Hi-C data to corresponding chromosome-length reference sequences. In addition to standard contact maps generated for all genomic positions across chromosomes the uploaded files include ACA (aggregate chromosome analysis) maps and phasing contact maps representing interactions between positions on the genome that contain heterogyzous SNPs. For more details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: wig: tracks showing the alignment frequency of Hi-C reads from read pairs that contain centromere repeat-specifc sequences.
Supplementary_files_format_and_content: bedpe: Files describing the tentative position of centromeres in chromosome-length genome assemblies, identified either by mapping repeat sequences associated with the centromere or via analysis of the contact map for evidence of centromere-to-telomere folding axis. For details see Hoencamp et al., Supplement.
Supplementary_files_format_and_content: vcf.gz: Variants (specifically SNPs) identivied by the DRAGEN variant calling pipeline from aligning Hi-C data to chromosome-length references generated via Hi-C-guided assembly and phased using the 3D-DNA Hi-C-guided phasing module.

organism: Notamacropus eugenii

source_name: blood

value: blood


title: GBM8_batch_3268


title: GBM8_batch_3263


title: 00078A16D3_IL


title: 00078A16DF_IL


title: GBM8_batch_3264


title: si-1

city: Jinan

first: Chunyan

last: Wang

state: Shandong

address: Wenhua West  No. 107,Lixia district

country: China

institute: Qilu Hospital of Shandong University

data_processing:
Sequenced reads were generated by base calling using the Illumina standard pipeline
Raw reads were first filtered to obtain clean reads using FASTX-Toolkit
High-quality reads were mapped to the human reference genome hg19 using TopHat software with default parameters
Alignment was independently performed for reads from each sample, and reads mapping to more than three genomic sites were discarded
Based on the value of reads per kilobase per million mapped reads, gene expression levels were determined using Cufflinks software
Genome_build: Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: Caski

growth_protocol: caski cells were cultured in phenol-red-free Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum

extract_protocol:
Total RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: caski cells were transfected with si-ER-α36 or NC for 48 h and treated with 1nM E2 for 24h

value: caski

value: squamous cervical cancer cell line

tag: transfection


title: 00078A0A43_IL


title: si-3

tag: cell type


title: NC-2

value: si-NC


title: NC-1


title: si-2


title: GBM8_batch_3259

tag: strain


title: GBM8_batch_3262


title: GBM8_batch_3258


title: GBM8_batch_3260


title: 00078A09B1_IL


title: GBM8_batch_3257


title: NC-3


title: 00078A0AEA_IL


title: 00078A096C_IL


title: 00078A087B_IL


title: MDA-MB-231-ML-2

city: Cambridge

first: Eric

middle: Scott

last: Lightcap

state: MA

address: 40 Landsdowne Street

institute: Takeda Pharmaceuticals

data_processing:
Raw fastq files were processed using the RNA-seq pipeline within Array Studio. Briefly, reads with bad qualities, or from IlluminaAdapters, Ercc, rRNA and tRNA were first filtered from raw data, the cleaned reads were then aligned to the genome through OmicSoft Aligner (OSA). The reads mapped to the exonic region were counted for each gene and then normalized into FPKM for the downstream analysis.
Genome_build: mouse NCBI genome build 38 and human NCBI genome build 37
Supplementary_files_format_and_content: The reads count and FPKM value of each gene in each sample are uploaded as the processed data files

source_name: cell line

growth_protocol: Cell lines THP-1, CT26, MC-38, B16F10, A20 were generated by re-plating at various densities from 80,000 to 200,000 cells per well in 12-well plates, in matched media supplemented with 10%FBS and/or 0.05mM β-Mercaptoethanol for 24 hour before treated with compounds. Cell lines HCT116, Colo205, MDA-MB-231 were generated by re-plating at 200,000 cells per well in 24-well plates, in matched media supplemented with 10%FBS for 24 hour before treated with compounds.

extract_protocol:
Cell pellets were collected after centrifuge, flash frozen on dry ice, and total RNA was extracted using RNeasy Mini Kit (Qiagen, 74104). Illumina TruSeq RNA Library Prep Kit (Cat#RS-122-2001) was used with 1 μg of total RNA for the construction of sequencing libraries.
RNAseq libraries were prepared for sequencing using standard Illumina protocols. Briefly, first polyA containing mRNA molecules were purified using oligo-dT attached magnetic beads from total RNA and then fragmented. The fragmented mRNA were reverse transcribed into first strand cDNA with reverse transcriptase and random primers. Then the RNA template were removed and replaced by synthesis of the second strand cDNA. Then the end of the cDNA fragments were repaired into blunt ends and adenylated at 3’ ends. Then the indexing adapters were ligated to the ends of cDNA fragments, and the DNA fragments with adapters on both ends were enriched and amplified by PCR. After clean-up, the PCR products go through quality control and quantitation to get the final library. Sequencing were run on Novaseq S4 platform: PE150.

treatment_protocol: THP-1, CT26, MC-38, B16F10, and A20 were seeded as triplets and treated with 1uM TAK-981 for 16 hours. HCT116, Colo205, and MDA-MB-231 were seeded as triplets and treated with 1uM TAK-981 or 1 uM ML-792 for 16 hours.

tag: treatment

tag: dosage

value: 1 uM

tag: timepoint


title: GBM8_batch_3254

extract_protocol:
For mice samples, collected brains were mechanically and enzymatically dissociated using a papain-based brain tumor dissociation kit (Miltenyi Biotec) supplemented with 0.1% typeI collagenase (Thermo Fisher Scientific) / PBS. The dissociated cells were first stained with calcein Blue AM (Thermo Fisher Scientific) and Zombie NIR (BioLegend) for 25 min at 4 ℃, and with anti-mouse CD16/32 (BD Biosciences) for 5min. After washing cells with ice-cold 1% BSA / PBS, the cells were then stained with a combination of following antibodies; PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences), phycoerythrin (PE) anti-mPdgfra (clone APA5, BioLegend), Allophycocyanin (APC) anti-mPodoplanin (clone 8.1.1, BioLegend), and APC anti-CD11b (clone M1/70, BioLegend), for 30 min at 4 ℃. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area, and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative. We sorted viable CD45 negative/ GFP positive or CD45 positive/ CD11b positive single-cells into 96-well plates containing 5uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol. Plates were frozen immediately after sorting and stored at -80 ℃ prior to whole transcriptome amplification, library preparation and sequencing.
Libraries from isolated single cells from mouse model were generated based on the Smart-seq2 protocol (Picelli 2014) with the following modifications. RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter) before oligo-dT primed reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 768 cells with unique barcodes were combined and sequenced with paired-end, 38-base reads, using a NextSeq 500 sequencer (Illumina). Comma-seperated file containing TPM values (transcript-per-million reads) for 6608 genes across 679 cells. For the cell lines, scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3’ Kit v2 and the 10x Chromium Controller (10x Genomics) according to the 10X Single Cell 3’ v2 protocol. The cell lines were processed as a pool that consisted of 6 total cell lines, 4 of which were used in this study (MGG23, MGG75, MGH143, MGG18). Two of the used cell lines, MGG23 and MGH143 were profiled again in a different pool. Briefly, we generated a single cell suspension of the pool in 0.04% PBS-BSA (95% viability) and loaded approximately 10,500 single cells to the Chromium Controller with a targeted recovery of 6,000 cells. Single cells, reagents and single gel beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized droplets and subjected to reverse transcription. Droplets were broken and the barcoded cDNAs were purified with DynaBeads and amplified by 12 cycles of PCR (98°C for 45 s; [98°C for 20 s, 67°C for 30 s, 72°C for 1 min] x 12; 72°C for 1 min). The amplified cDNA was fragmented, end-repaired, ligated with index adaptors, and size-selected with clean-ups between each step using the SPRIselect Reagent Kit (Beckman Coulter). Quality control of the resulting barcoded libraries was performed with the Agilent TapeStation and by PCR with primers specific to the P5 and P7 sequence (NEBNext Library Quant Kit for Illumina, New England Biolabs).
single cell RNA-seq


title: MDA-MB-231-ML-3


title: GBM8_batch_3261


title: 00078A0255_IL

tag: Sex


title: 00078A07A2_IL


title: MDA-MB-231-TAK-3


title: MDA-MB-231-TAK-2


title: GBM8_batch_3256


title: MDA-MB-231-ML-1


title: GBM8_batch_3255


title: 00078A02CB_IL


title: GBM8_batch_3253


title: 00078A0246_IL


title: 00078A02DF_IL


title: MDA-MB-231-veh-3

value: DMSO


title: GBM8_batch_3249


title: 00078A0224_IL


title: GBM8_batch_3252


title: 00078A0244_IL


title: GBM8_batch_3251


title: colo205-ML-2


title: 00078A022D_IL


title: MDA-MB-231-veh-2


title: GBM8_batch_3250


title: 00078A01DB_IL


title: GBM8_batch_3247


title: MDA-MB-231-TAK-1


title: 00078A021A_IL


title: 00078A01FE_IL


title: 00078A0215_IL

description: Removed: too many zero counts


title: colo205-TAK-1


title: colo205-ML-1


title: colo205-TAK-3


title: 00078A01A6_IL


title: MDA-MB-231-veh-1


title: colo205-ML-3


title: GBM8_batch_3245


title: colo205-TAK-2


title: GBM8_batch_3244


title: GBM8_batch_3248


title: 00078A0189_IL


title: 00078A01C0_IL


title: GBM8_batch_3246


title: HCT116-ML-3


title: colo205-veh-3


title: 00078A0139_IL


title: GBM8_batch_3242


title: GBM8_batch_3241


title: GBM8_batch_3240


title: 00078A0166_IL


title: HCT116-ML-2


title: colo205-veh-2


title: 00078A0138_IL


title: 00078A01D8_IL


title: GBM8_batch_3239


title: HCT116-ML-1


title: GBM8_batch_3243


title: GBM8_batch_3238


title: GBM8_batch_3237


title: HCT116-TAK-1


title: 00078A0085_IL


title: 00078A0058_IL

description: Removed: expression outlier


title: GBM8_batch_3234


title: GBM8_batch_3235


title: GBM8_batch_3236


title: 00078A00BF_IL


title: 00078A00AC_IL


title: HCT116-TAK-3


title: 00078A0127_IL


title: HCT116-veh-3


title: HCT116-TAK-2


title: colo205-veh-1


title: 00078A0041_IL


title: 00078A002C_IL


title: HCT116-veh-1


title: THP-1-TAK-3


title: THP-1-TAK-2


title: GBM8_batch_3233


title: 000789FFF9_IL


title: GBM8_batch_3232


title: THP-1-TAK-1


title: GBM8_batch_3231


title: GBM8_batch_3230


title: GBM8_batch_3229


title: 000789FFF8_IL

description: Removed: genotype mismatch


title: GBM8_batch_3228


title: GBM8_batch_3227


title: THP-1-veh-6


title: GBM8_batch_3225


title: 000789FF6E_IL


title: GBM8_batch_3224


title: hiPSCs_D20X_HP_repC

city: Leuven

first: Marino

last: Caruso

address: Herestraat 49

department: Oncology

country: Belgium

institute: KULeuven

description: High-polysome (HP) RNA extract

data_processing:
Library strategy: polysome profiling
 Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument.
Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.
sequence read were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36.
The trimmed reads will be mapped to the Homo sapiens reference genome available on ENSEMBL using the STAR aligner v.2.5.2b.
Unique gene hit counts will be calculated by using feature Counts from the Subread package v.1.5.2. Only unique reads that fall within exon regions will be counted.
Supplementary_files_format_and_content: Table of gene hit counts for each replicate

source_name: HC3X hiPSCs-derived Hepatocyte-like cells

growth_protocol: HC3X iPSCs were cultured on hESCs qualified-Matrigel coated vessels in mTESR PLUS medium supplemented with 1% penicillin-streptomycin. Cells were passaged every 5-6 days by accutase detachment and plated at a 1/10 dilution. Cells were replated in medium supplemented with 10uM ROCK inhibitor (Y27632) for the first 24h.

extract_protocol:
ReliaPrep RNA extraction (Promega Z6010)
NEBNext Ultra II Directional RNA Library Prep Kit

treatment_protocol:
STD and HC3X in vitro hepatogenic differentiation
TotRNA: Cell lysis
High-Polysome (HP) RNA: Cell lysis, sucrose density fractionation, selection of high-polysome containing fractions

value: HC3X hiPSCs

value: D20 HC3X hepatogenic differentiation

tag: replicate

value: C