title: Bos taurus, m6A-Seq, post_4_IP RNA


title: F4-AsIII 0.1 uM 48h

status: Public on Sep 30 2024

type: SRA

city: Bergen

first: Kai

middle: Kristoffer

last: Lie

state: Hordaland

address: Pobox 2029 Nordnes

department: Feed Safety

country: Norway

institute: Institute of Marine research

description: Hepatocyte isolated from individual fish were exposed to either arsenosugar (AsSug) at different concentrations in addition to control (AsSugControl) or arsenite (AsIII) at different concentrations in addition to control on individual plates for each compound.

library_source: transcriptomic

data_processing:
raw sequencing reads were mapped to the current Atlantic salmon genome (GCF_905237065) downloaded from the NCBI genome database using the Hisat2 mapper and counted using FeatureCounts as part of the Subread package. Raw counts were further normalized using DESeq2 (1.40.2) Bioconductor package (V3.17), features that had lower count than 10 reads were filtered prior to normalization. Differentially expressed genes (DEGs) were determined using Wald test and corrected for multiple testing using the Benjamini and Hochberg test, as implemented in the DESeq2 package. Differentially expressed genes were used for further downstream pathway analysis. Due to the high number of differentially expressed genes in AsIII exposed cells, a 2-fold differential expression cut of was applied to these samples. Due to limited tools and databases for biological function of non-model species, DEG feature gene names were converted to human ortholog gene names using the BioDBnet database (https://biodbnet-abcc.ncifcrf.gov/db/dbOrtho.php) prior to pathway analysis. Pathway analysis were conducted using the open source Metascape software (https://metascape.org)
Assembly: GCF_905237065
Supplementary files format and content: The processed datafile contain a matrix of normalized gene counts in txt format.

molecule: total RNA

organism: Salmo salar

source_name: Liver

growth_protocol: Hepatocytes were isolated from six male Atlantic salmon (447–572g) with a two-step perfusion method. Briefly, a peristaltic pump (Miniplus 3, Gilson, USA) was used to clear the liver for blood (4 mL/min) via the hepatic vein followed by digestion with collagenase (1 mg/mL). When the digestion of the liver was complete, the organ was removed from the fish. The hepatocytes were gently shaken in PBS by using tweezers. Subsequently, the cell suspensions were filtered (100 μm nylon gauze) and the cells collected by centrifugation (50 × g for 5 min). The hepatocytes were washed twice with PBS. The cells were collected by centrifugation (50 × g for 5 min). The supernatant was decanted, and the final cell pellet was resuspended in L-15 medium (without phenol red) containing 10% fetal bovine serum (Sigma Aldrich), 1% glutamax (Invitrogen, Norway) and 1% penicillin-streptomycin-amphotericin (10000 units/mL potassium penicillin, 10000 μg/mL streptomycin sulphate and 25 μg/mL amphotericin B) (Lonzo, Medprobe, Oslo, Norway). Cell viability was determined following the manufacturer’s protocol of Trypan Blue exclusion methodology (Lonzo, Medprobe, Oslo, Norway). The different cell suspensions used in this study had hepatocyte cell viability >90%. Fish handling was approved by the Norwegian Animal Research Authority (FOTS ID, 19351). The hepatocytes were plated on 2 μg/cm2 laminin (L2020, Sigma-Aldrich, Oslo, Norway) coated 3D 6-well Alvetex® scaffolds (200 μm cross-linked polystyrene membranes, 42 μm mean void size, Reprocell, Glasgow, United Kingdom) in 6 well culture plates (Falcon, Corning, VWR, Bergen, Norway). The following cell concentrations were used; 7.2 × 106 cells per scaffold in 6-well plates (in 10 mL complete L15 medium) and 0.2 × 106 cells per well in 96-well plates (in 0.200 mL or 0.150 mL complete L-15 medium in 2D and 3D, respectively). the hepatocytes were kept at 10 °C in a sterile incubator without additional O2/CO2 (Sanyo, CFC FREE, Etten Leur, Netherland).

extract_protocol:
Hepatocytes for RNA extraction and sequencing were seeded on 6-well Alvetex® scaffolds. After 48 h exposure to arsenite, arsenate and arsenobetaine, 2 mL of cell medium from each well was taken and kept at -20ºC until further analysis (see section 2.6). The rest of the cell medium was discarded. Retrieval of cells from Alvetex® scaffolds for this analysis was performed according to manufacturer’s description (see protocol for retrieval of cells from Alvetex® scaffold's for metabolomic analysis). In summary, the well inserts with the hepatocyte cells were transferred to new 6-well plates. The cells were washed with PBS (1 mL) which was removed from the well before the next step. The Alvetex Scaffold membrane was removed with the aid of flat-ended forceps. To break down the cells, introduce 600 µL of the Qiagen RNeasy® kit lysis buffer RLT directly to the well where is Alvetex Scaffold membrane was left. Allow the plate on a rotating platform (100 rpm) for 10 min at room temperature. Conclude the process by adding 600 µL of 70% ethanol to the now-homogenized lysate. Ensure a proper mix by pipetting the combination up and down around 10 times. The cell suspension was transferred to centrifugation tubes and centrifuged at 1000×g for 5 min at 4 °C. Cell pellets were flash frozen and stored at −80 °C until RNA extraction was performed.Total RNA was extracted from Atlantic salmon Primary hepatocytes using a RNeasy Plus Mini Kit (Qiagen, Crawley, UK) as previously described (Søderstrøm et al 2023). Total RNA purity was evaluated using a nanodrop NanoDrop™ (Thermo Fisher Scientific™, MA, USA) Samples had a 260/230 and 260/280 ratio of 2.2 ± 0.1 and 2.09 ± 0.03 respectively. Integrity of RNA for each sample was investigated using RNA 6000 Nano LabChips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Although the integrity of the majority of the samples were assessed as good (RIN = 9.7 ± 0.12, N = 48), RNA from cells exposed to 250 µM AsSug showed a reduction in integrity (RIN 5.3 ± 0.5, N = 6).
RNAseq of arsenite and arsenosugar 328 samples was conducted by a commercial company (Azenta Life Sciences, Leipzig, Germany). RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated using DNA Kit on the Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA). The sequencing libraries were multiplexed and clustered onto a flow-cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.

treatment_protocol: Aqueous solutions of arsenite (Spectrascan, 1000ug/mL) were purchased from Teknolab (Ski, Norway). Arsenosugar glycerol was gently given by Jack Creed of the Environmental Protection Agency (EPA) National Exposure Research Laboratory in Cincinnati, OH, USA. Arsenite and arsenosugar glycerol used in the exposure experiments were dissolved individually to constitute a 1 mM stock solution from which dilutions were made. The stock solutions were diluted in Leibovitz L-15 medium w/o phenol red supplemented with 10% FBS and 1% antibiotic antimycotic solution. The primary cells were cultured for 36–40 h prior to chemical exposure. Liver cells from six fish (N = 6) were exposed for 24 h and 48 h to establish dose-response curves for arsenite (0.1, 1, 3, 10 and 30 μM), arsenate (10, 50, 100 and 500 μM), arsenobetaine (10, 50, 100 and 500 μM) and for arsenosugar glycerol (0.5, 5, 10, 50, 50, 100, 250 μM). The concentrations used were selected based on earlier in vitro toxicity assessments

tag: tissue

value: Liver

tag: cell line

value: Primary cells

tag: cell type

value: Primary hepatocytes

tag: FISH number

tag: treatment


title: repRNA_v4_post_day1_rep3

city: Beijing

first: Yihan

last: Lin

address: No.5 Yiheyuan Road, Haidian

country: China

institute: Peking University

data_processing:
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: Coding sequence of EGFP
Supplementary files format and content: The processed files in csv format contain the total mutation frequency for each sample.

organism: Mesocricetus auratus

source_name: kidney

growth_protocol: BHK-21 cells were cultivated in Minimum Essential Medium with Earle's Balanced Salts (MEM/EBSS) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/ml streptomycin, and 1% MEM Non-Essential Amino Acids Solution (Gibco, #11140050) in a humidified 37℃ incubator with 5% CO2.

extract_protocol:
The cells were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA or IVT RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
EGFP region was amplified from the cDNA or DNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and specific primers(For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages.

value: kidney

value: fibroblast cells

tag: genotype

value: repRNA-v4 based

value: no


title: Bos taurus, m6A-Seq, post_4_input RNA

city: Changchun

first: Xiaorui

last: Yang

address: Xincheng Street

institute: Jilin Agricultural University

data_processing:
Base calling software: BCL convert
Data filter: fastp(0.19.4)，remove adapters, repeat sequences and low quality sequences
reads alignment : HISAT2(2.0.4)
Peak calling diffpeak : R package: exomePeak (1.5.0)
Peak annotation : ANNOVAR
Transcript assembly and quantification : StringTie (2.1.2)
Differential Expression Analysis :R package: edgeR(4.1)
Motif: HOMER(4.10)
Assembly: BOVINE(ensemble V107)
Supplementary files format and content: bigWig
Library strategy: m6A-seq

molecule: polyA RNA

organism: Bos taurus

source_name: pituitary

extract_protocol:
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification.
the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. The cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol

value: pituitary


title: repRNA_v4_post_day1_rep2


title: Leaf of Rhodendron ‘xiaotaohong’, 40℃, melatonin-applied, rep2

city: Zhuji

first: Yanxia

last: Xu

state: Zhejiang

address: 66 Puyang Road

institute: Jiyang College, Zhejiang A&F University

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/, version:cutadapt-1.9).
We aligned reads of all samples to the Rhododendron reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/, version:hisat2-2.0.4) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome. HISAT2 allows multiple alignments per read (up to 20 by default) and a maximum of two mismatch when mapping the reads to the reference. HISAT2 build a database of potential splice junctions and confirms these by comparing the previously unmapped reads against the database of putative junctions.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/, version:stringtie-1.3.4d) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software (http://ccb.jhu.edu/software/stringtie/gffcompare.shtml, version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Assembly: Rhododendron simsii
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

organism: Rhododendron

source_name: leaf

extract_protocol:
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp.

tag: variety

value: xiaotaohong

value: leaf


title: Bos taurus, m6A-Seq, post_3_IP RNA


title: Leaf of Rhodendron ‘xiaotaohong’, 35℃, melatonin-applied, rep3


title: repRNA_v4_post_day1_rep1


title: Leaf of Rhodendron ‘xiaotaohong’, 40℃, normal, rep2


title: F4-AsIII Control 48h


title: F3-AsIII 3 uM 48h


title: Bos taurus, m6A-Seq, post_3_input RNA


title: Leaf of Rhodendron ‘xiaotaohong’, 40℃, normal, rep1


title: Bos taurus, m6A-Seq, post_2_IP RNA


title: repRNA_v4_post_day14_rep3


title: Leaf of Rhodendron ‘xiaotaohong’, 35℃, normal, rep3


title: F3-AsIII 0.1 uM 48h


title: Bos taurus, m6A-Seq, post_2_input RNA


title: repRNA_v4_post_day14_rep2


title: Bull sperm 4

city: Madrid

first: Alfonso

last: Gutiérrez-Adán

address: Av. Puerta de Hierro 28

department: Animal Reproduction

country: Spain

institute: INIA

data_processing:
Raw reads were processed and mapped using CLC Genomics Workbench
Bowtie2 was used to asses the presence of rRNA and filter the reads
Unmapped reads were aligned to the RefsSeq bacterial genome library using Kraken2 and Bracken was used to improve abundance estimation
Assembly: Bos Taurus ARS-UCD1.3 and Equus Caballus EquCab3.0
Supplementary files format and content: Excel files include bacterial abundance for each sample

source_name: Spermatozoa

extract_protocol:
Total RNA was isolated from sperm using TRIzol reagent, treated with Dnase for 15 min and purified by phenol:chloroform extraction
RNA libraries were prepared using the NEBNext UltraTM RNA Library Prep Kit

value: Spermatozoa

value: Fertile


title: F3-AsIII 1 uM 48h


title: F3-AsIII Control 48h


title: Leaf of Rhodendron ‘xiaotaohong’, 35℃, normal, rep2


title: Leaf of Rhodendron ‘xiaotaohong’, 25℃, normal, rep3


title: F1-AsIII Control 48h


title: Bull sperm 2


title: Bull sperm 3


title: repRNA_v4_post_day14_rep1


title: Leaf of Rhodendron ‘xiaotaohong’, 40℃, normal, rep3


title: Leaf of Rhodendron ‘xiaotaohong’, 35℃, normal, rep1


title: Leaf of Rhodendron ‘xiaotaohong’, 25℃, normal, rep2


title: F1-AsSug 250 uM 48h


title: F2-AsSug 250 uM 48h


title: Bull sperm 1


title: F6-AsSug 250 uM 48h


title: repRNA_v4_IVTRNA_rep2


title: repRNA_v4_plasmid_rep1


title: repRNA_v4_plasmid_rep3


title: repRNA_v4_plasmid_rep2


title: F4-AsSug 250 uM 48h


title: repRNA_v4_IVTRNA_rep3


title: Leaf of Rhodendron ‘xiaotaohong’, 25℃, normal, rep1


title: F3-AsSug 250 uM 48h


title: TetR-VP48-Post_selection_728-d1

data_processing:
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: Coding sequence of TetR-VP48, or PadR
Supplementary files format and content: The processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
Target genes (e.g., TetR-VP48, PadR) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: To evolve PadR, medium was replaced with fresh medium containing 5 μg/ml of puromycin and 10 µg/ml blasticidin 24 h post-electroporation. After 4 d of selection, two million cells were transferred into a new 10-cm culture dish and subjected to consecutive two-day treatment with molnupiravir (10 µM, with daily media changes) to induce RNA mutation. Upon completion of molnupiravir treatment, we adopted different selection/screening strategies according to the specific evolution objectives (i.e., one to evolve ligand-resistant mutants and the other to evolve mutants with enhanced target gene activation in the absence of ligand). To decrease or reverse ligand sensitivity, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the presence of the ligand (200 µM sodium ferulate). In the initial phase of selection, we gradually escalated the concentration of puromycin to inhibit excessive cell growth in the 10-cm culture dish. By enhance the puromycin concentration (up to 60 µg/ml) during mid-screening (e.g., approximately on day 7), around 99% of cells in the dish were effectively eliminated (Approximately 104 to 105 cells in the dish survived in this ‘bottleneck’ stage). This selection was then sustained for another several days. Upon completion of the evolution, cells were collected for RNA extraction and mutation analysis. To enhance the response dynamic range of PadR, we employed an evolutionary approach by selecting replicative RNAs containing VP64-PadR with increasing puromycin concentration in the absence of the ligand (sodium ferulate). We employed a similar approach to gradually enhance the puromycin concentration. However, due to excessive PadR activation activity in the absence of sodium ferulate, we encountered difficulties in eliminating cells by enhancing puromycin concentration (up to 80 µg/ml). Consequently, at the conclusion of the screening period, we utilized flow sorting to isolate approximately the top 1% mCherry-expressing cells (Approximately 105 cells in each dish) from the remaining population for RNA extraction and mutation analysis. To evolve TetR, we employed a similar protocol used in the evolution of PadR to evolve doxycycline-resistant TetR mutants in the presence of 1 µg/ml doxycycline and continuous treatment of puromycin (up to 80 µg/ml).

value: molnupiravir


title: F6-AsSug 50 uM 48h


title: Adult ALI, RSV, rep1

city: Boston

first: Caiqi

last: Zhao

address: 55 Fruit St

country: USA

institute: MGH

description:
10x Genomics
Series supplementary file: features.tsv.gz

library_source: transcriptomic single cell

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1.
Assembly: hg38
Supplementary files format and content: Tab-separated values files and matrix files

molecule: nuclear RNA

organism: Homo sapiens

source_name: ALI culture of tracheal aspirate derived airway basal stem cells

extract_protocol:
For one 24-well ALI culture insert, the membrane was removed from the insert and treated with 500 µl of Accutase (Cat#A1110501, Thermo Fisher Scientific) with EDTA and EGTA (5 mM each) for 30 minutes at 37℃ on a sharker. After treatment, cells were dissociated by titrating and cell pellets were collected by centrifugation. After 1X PBS wash to remove the remaining Accutase, cells were resuspended in nuclear extraction buffer according to manufacturer’s instruction (10X Genomics, protocol CG000124).
The RNA library was constructed using 10X Single Cell 3’ GEX and libraries were sequenced on Illumina NovaSeqS4 (150x150 Paired-End).
single nuclei RNA sequencing

tag: age

value: Adult

value: RSV


title: Adult ALI, Mock, rep1

value: ALI culture of tracheal aspirate derived airway basal stem cells

value: Mock


title: repRNA_v4_IVTRNA_rep1


title: PadR_evolution_with_ligand_793


title: Neonatal ALI, Mock, rep1

value: Neonatal


title: F1_AsB_100uM

description:
F1_154
Hepatocyte isolated from individual fish were exposed to either arsenosugar (AsSug) at different concentrations in addition to control (AsSugControl) or arsenite (AsIII) at different concentrations in addition to control on individual plates for each compound.

extract_protocol:
Hepatocytes for RNA extraction and sequencing were seeded on 6-well Alvetex® scaffolds. After 48 h exposure to arsenite, arsenate and arsenobetaine, 2 mL of cell medium from each well was taken and kept at -20ºC until further analysis (see section 2.6). The rest of the cell medium was discarded. Retrieval of cells from Alvetex® scaffolds for this analysis was performed according to manufacturer’s description (see protocol for retrieval of cells from Alvetex® scaffold's for metabolomic analysis). In summary, the well inserts with the hepatocyte cells were transferred to new 6-well plates. The cells were washed with PBS (1 mL) which was removed from the well before the next step. The Alvetex Scaffold membrane was removed with the aid of flat-ended forceps. To break down the cells, introduce 600 µL of the Qiagen RNeasy® kit lysis buffer RLT directly to the well where is Alvetex Scaffold membrane was left. Allow the plate on a rotating platform (100 rpm) for 10 min at room temperature. Conclude the process by adding 600 µL of 70% ethanol to the now-homogenized lysate. Ensure a proper mix by pipetting the combination up and down around 10 times. The cell suspension was transferred to centrifugation tubes and centrifuged at 1000×g for 5 min at 4 °C. Cell pellets were flash frozen and stored at −80 °C until RNA extraction was performed.Total RNA was extracted from Atlantic salmon Primary hepatocytes using a RNeasy Plus Mini Kit (Qiagen, Crawley, UK) as previously described (Søderstrøm et al 2023). Total RNA purity was evaluated using a nanodrop NanoDrop™ (Thermo Fisher Scientific™, MA, USA) Samples had a 260/230 and 260/280 ratio of 2.2 ± 0.1 and 2.09 ± 0.03 respectively. Integrity of RNA for each sample was investigated using RNA 6000 Nano LabChips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Although the integrity of the majority of the samples were assessed as good (RIN = 9.7 ± 0.12, N = 48), RNA from cells exposed to 250 µM AsSug showed a reduction in integrity (RIN 5.3 ± 0.5, N = 6).
Total RNA samples of arsenate and arsenobetaine were sent to Novogene Europe, Cambridge, UK, for cDNA library preparation and sequencing using the Illumina NovaSeq 6000 platform for 150 bp paired end reads. Enriched mRNA was prepared using Oligo(dT beads). cDNA libraries were prepared following manufacturer's instructions per the Novogen pipeline (Novogene Europe, Cambridge, UK)

value: Liver

value: Primary cells


title: WT_LPS_16h_5

city: Indianapolis

first: Takashi

last: Hato

state: IN

address: 950 W. Walnut St. R2 202

department: Medicine

institute: Indiana University

description:
IB20
BambuOutputcounts_gene.txt

data_processing:
The basecalling was done using Guppy basecaller: guppy_basecaller --compress_fastq --fast5_out -i ./fast5_pass/ -s ./fastq/ --device 'auto' --num_callers 1 --flowcell "FLO-MIN106" --kit "SQK-DCS109"
Mapping was carried out using Minimap2: minimap2 -a -L -t 12 -x splice --junc-bed Mus_musculus.GRCm38.101.bed --MD mm10-ont.mmi ./fastq/*.fastq.gz 2> ./minimap2.err > ./SAM/$sample.sam
Transcript-level analysis was performed using Bambu
Assembly: mm10
Supplementary files format and content: counts table in text format

organism: Mus musculus

extract_protocol:
Snap-frozen mouse kidney tissue was homogenized in 800 µl of Tri Reagent using a Minilys tissue homogenizer at the highest speed for 45 seconds and then incubated for 5 minutes. Total RNA was extracted from 600 µl of the supernatant using the Direct-zol RNA miniprep Plus (Zymo Research), including on-column DNase I digestion. The RNA was eluted in 100 µl of water and subjected to mRNA polyA enrichment using the Dynabeads mRNA DIRECT Micro kit (Thermo Fisher 61021). After two rounds of washing and enrichment of mRNA following the manufacture’s protocol, polyA+ mRNA was eluted in 10 µl of water.
We proceeded with approximately 200 ng of polyA+ mRNA as the starting material. Reverse transcription and strand-switching was done using Maxima H Minus Reverse Transcriptase following the Nanopore protocol (SQK-DCS109). After the second strand synthesis, the RNA concentration was generally 20-30 ng/µl as measured by NanoDrop. Following end-prep, adapter ligation, and AMPure XP bead binding, the yield was generally 6-10 ng/µl as measured by Qubit. Sequencing was conducted on R9.4.1 flow cells using the GridIon platform.

value: kidney

value: LPS 4 mg/kg iv 16 h


title: WT_LPS_28h_3

description:
IB12
BambuOutputcounts_gene.txt

value: LPS 4 mg/kg iv 28 h


title: F6-AsSug 10 uM 48h

value: Primary hepatocytes


title: MEK1_day1_rep3_784-3

data_processing:
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: Coding sequence of MEK1 from H. sapiens
Supplementary files format and content: The processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
Target gene (e.g., MEK1) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: Medium was replaced with fresh medium containing 5 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 days of selection by puromycin (i.e., on Day 5), approximately 2 million of cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using a concentration of 2 μM molnupiravir. On Day 7, the cells in each dish were approaching 90% confluence (~ 107 cells per dish). Therapeutic drug (i.e., 2-5 μM cobimetinib, 5-10 μM trametinib, or 50-100 μM selumetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One week later (i.e., Day 14), approximately 105 cells successfully survived and adapted to the environment with inhibitor. The cells were collected for total RNA extraction, sequencing, and mutation analysis.

value: fibroblast cells

value: repRNA-v4 based

value: No


title: F5-AsSug 250 uM 48h


title: WT_LPS_28h_2

description:
IB11
BambuOutputcounts_gene.txt


title: WT_LPS_28h_1

description:
IB10
BambuOutputcounts_gene.txt


title: MEK1_day1_rep1_784-1


title: MEK1_day1_rep2_784-2


title: Whole_blood_LRFI_30_4381_0_13

type: RNA

city: Castanet-Tolosan

first: Laurence

last: Liaubet

address: 24 chemin de Borde Rouge

department: Animal genetics

country: France

institute: INRAE

description: castrated

hyb_protocol: 600 ng of Cy3-labelled cRNA (specific activity >8 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse GE microarray (8X60K, Design 028005) enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min), Wash buffer 2 (Agilent Technologies, 37°C, 1 min).

scan_protocol: Slides were scanned immediately after washing on a Agilent G2505C Microarray Scanner with Agilent Scan Control A.8.5.1 software.

data_processing: The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol GE1_1010_Sep10 and Grid: 037880_D_F_2012). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package. Probes were filtered and kept if they had a good signal in more than 75% of the samples in at least one experimental group. Using this filter, 32924 probes were selected from a total of 61625, or 53.4%,  normalized by the median, and log2 transformed. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. A chip as batch effect was corrected with using the ComBat procedure from the sva package.  The resulting matrix has 32924 rows each corresponding to a unique ProbeName (provided as data Matrix) and 75 columns (pigs x day +/- hour).

organism: Sus scrofa

source_name: Whole blood

label_protocol: For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy purification (QIAGEN). Dye incorporation and cRNA yield were checked with the Biospec nano spectrophotometer (Shimadzu).

growth_protocol: The pigs were raised in selection facilities (INRAE, GenESI, Le Magneraud, France, doi : 10.15454/1.5572415481185847E12), then transferred to the experimental facilities of INRAE in Saint-Gilles (Pig Physiology and Phenotyping Experimental Facilit, doi 10.15454/1.5573932732039927E12) at approximately 80 days of age (i.e., 40 kg of BW).

extract_protocol: Total RNA was extracted according to the manufacturer's recommendations (PAXgene Blood RNA Kit, Qiagen) and the extracted total RNA was eluted in 40 µl of buffer BR5 and stored at -80°C. Quantification was performed using a Nanodrop to determine the concentration of each purification and the 260/230, 260/280 ratios. Quality was checked by electrophoresis on 0,8% agarose gel. RNA Integrity Numbers (RINs) were determined using a 2100 Bioanalyzer Instrument (Agilent).

treatment_protocol: Whole blood RNA were collected on PAXgene Blood RNA Tubes (PreAnalytiX) and were conserved at -20°C until extraction.

tag: repetition

tag: breed

value: LRFI

tag: pen

tag: temperature

tag: gender

value: neuter


title: WT_LPS_16h_3

description:
IB17
BambuOutputcounts_gene.txt


title: Neonatal ALI, RSV, rep1


title: Whole_blood_HRFI_24_4447_-5

description: castrated

value: HRFI


title: Whole_blood_HRFI_30_4325_2

description: castrated


title: Whole_blood_HRFI_24_4462_0_9

description: castrated


title: Whole_blood_HRFI_24_3394_-5

description: castrated


title: Whole_blood_HRFI_30_4323_0_18

description: castrated


title: F6-SugControl 48h

value: AsSugControl


title: F5-AsSug 5 uM 48h


title: MEK1_day14_863_Sel_rep1

value: molnupiravir


title: Whole_blood_LRFI_30_4307_0_18

description: castrated


title: PadR_evolution_without_ligand_792


title: Whole_blood_HRFI_30_3386_1

description: castrated


title: Whole_blood_LRFI_24_3371_-5

description: castrated


title: F5-AsSug 50 uM 48h


title: MEK1_day14_865_Sel_rep3


title: F5-SugControl 48h


title: MEK1_day14_829_Tra_rep3


title: WT_LPS_16h_4

description:
IB19
BambuOutputcounts_gene.txt


title: F6-AsSug 5 uM 48h


title: MEK1_day14_828_Tra_rep2


title: F5-AsSug 10 uM 48h


title: F4-AsSug 50 uM 48h


title: Whole_blood_LRFI_30_4370_1

description: castrated


title: F4-AsSug 10 uM 48h


title: F4-AsSug 5 uM 48h


title: WT_LPS_04h_2

description:
IB8
BambuOutputcounts_gene.txt

value: LPS 4 mg/kg iv 4 h


title: WT_LPS_04h_1

description:
IB7
BambuOutputcounts_gene.txt


title: F4-SugControl 48h


title: MEK1_day14_864_Sel_rep2


title: Whole_blood_HRFI_30_4462_0_13

description: castrated


title: F3-AsSug 50 uM 48h


title: MEK1_day14_824-Cob_rep1


title: WT_LPS_16h_2

description:
IB4
BambuOutputcounts_gene.txt


title: WT_LPS_16h_1

description:
IB1-3
BambuOutputcounts_gene.txt


title: WT_LPS_04h_3

description:
IB9
BambuOutputcounts_gene.txt


title: MEK1_day14_827_Tra_rep1


title: Whole_blood_LRFI_30_3357_0_18

description: castrated


title: Whole_blood_HRFI_30_4447_2

description: castrated


title: WT_ctrl_3

description:
IB22
BambuOutputcounts_gene.txt

value: Saline


title: MEK1_day14_826_Cob_rep3


title: MEK1_day14_825_Cob_rep2


title: Whole_blood_LRFI_24_4420_-5

description: castrated


title: Whole_blood_LRFI_30_3371_0_13

description: castrated


title: WT_LPS_01h_3

description:
IB15
BambuOutputcounts_gene.txt

value: LPS 4 mg/kg iv 1 h


title: WT_LPS_01h_2

description:
IB14
BambuOutputcounts_gene.txt


title: Whole_blood_LRFI_24_4420_0_9

description: castrated


title: KRAS_WT_day14_rep3_820

data_processing:
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: CDS of KRAS or KRAS (S17N) from H. sapiens
Supplementary files format and content: The processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
Target genes (e.g., KRAS WT or KRAS S17N) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: Medium was replaced with fresh medium containing 10 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 d of selection (i.e., Day 5), approximately 2 million cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using molnupiravir (2 μM). When the plate reached approximately 90% confluence, the cells were consistently subcultured at a 1:5 ratio and the medium was daily replenished with 10 μg/mL puromycin and 2 μM molnupiravir treatment. After another 9 d of culture, cells were harvested for total RNA extraction, sequencing, and mutation analysis.


title: F2-AsSug 10 uM 48h

value: Liver


title: KRAS_WT_day14_rep2_819


title: F3-AsSug 10 uM 48h

value: Primary cells


title: KRAS_WT_day1_rep3_779-3

value: kidney


title: F3-AsSug 5 uM 48h


title: Whole_blood_HRFI_30_4447_1

description: castrated


title: RNA_T_UNC0224_1uM_2_rep1

first: Mohamad Ali

last: Najia

state: MA

address: 1 Blackfan Circle

department: Hematology/Oncology

institute: Boston Children's Hospital

description:
Bio rep 2, tech rep 1
T_UNC_2

data_processing:
RNA-Seq paired-end reads were pseudo-aligned with kallisto (Bray et al. 2016) to the GRCh37/hg19 reference transcriptome. Transcript-level abundance estimates were imported into R with the tximport package and constructed into a gene-summarized count and abundance matrices for all samples. Differential gene expression analysis was performed using DESeq2 (Love, Huber, and Anders 2014) on the estimated count matrix. Differentially expressed genes were defined by a abs(log2-fold change) > 1 and FDR < 0.05.
Assembly: hg19
Supplementary files format and content: Tab-separated file of the kallisto counts or TPM abundance matrix

source_name: 1157.2 iPS cell line

growth_protocol: Human 1157.2 iPSCs were differentiated into CD34+ HE cells using a previous described protocol (Ditadi et al., 2015). Briefly, iPSC colonies cultured on mouse embryonic fibroblast (MEF) feeder cells (Gibico, A34181) were scraped and transferred to ultra-low attachment plates and cultured with aggregation media contains BMP4 (10ng/ml, day 0-2), bFGF (5ng/ml, day 1-2), CHIR99021 (3μM, day 2), and SB431542 (6μM, day2) to allow EB formation. After 4 days, EBs were cultured with StemPro-34 media (Gibco, 10639011) supplemented with VEGF (15ng/ml, day3-8), bFGF (5ng/ml, day3-8), SCF (50ng/ml, day 6-8), EPO (day6-8), IL-6 (day 6-8), IL-11 (day 6-8), and IGF-1 (day 6-8). On day 8, EBs were dissociated using EB Dissociation Kit (Miltenyi, 130096348), and HE cells were isolated by magnetic-activated cell sorting (MSCS) using the human CD34 Microbead Kit (Miltenyi, 130046702). iPSC-derived HSPCs were differentiated in to T cells using a previously reported protocol (Jing et al., 2022). Briefly, HSPCs or HEs were seeded on non-tissue culture treated plates coated with 10μg/ml recombinant human DLL4-Fc protein (Life Technologies, A42510) and 2 μg/ml VCAM-1 (Shukla et al., 2017) (R&D, 862VC100) and cultured in SFEMII (StemCell Tech, 09605) media supplemented with 10% BIT serum substitute (StemCell Tech, 09500), 2mM L-Glutamine (Gibco, 35050061), 1mM sodium pyruvate (Gibco, 11360070), 50μg/ml 2-phospho-L-ascorbic acid (Huijskens et al., 2014) (sigma, 49752), 55μM 2-Mercaptoethanol (Gibco, 21985023), 1mM None-essential amino acids (Gibco, 11140050), 1% penicillin-streptomycin (Corning, 30002CI), 30ng/ml SCF, 20ng/ml FLT3, 30ng/ml IL-7, 5ng/ml IL-3 (first week only), and 5ng/ml TPO for 2 weeks to induced the differentiation into proT cells. After 2 weeks, proT cells were replated into new DLL4-coated plates and cultured in the presence of 20ng/ml FLT3 and 15ng/ml IL-7 till week 4, followed by 1 week of treatment with CD3/CD28 T activator (StemCell Tech, 10971) and 5 ng/ml IL-15 to induced SP T cells. During T cell differentiation, half media changes were conducted every 2-3 days.

extract_protocol:
DAPI-CD45+ cells were isolated by FACS and at least 5,000 cells/sample were used for RNA-Seq
At least two technical replicates were prepared per sample using the SMART-Seq2 protocol (Picelli et al. 2014), with some modifications. Briefly, RNA was purified from cellular lysate using 2.2X RNA SPRI beads (Beckman Coulter cat. no. A63987) then immediately reverse transcribed in the presence of a template switching oligo (Exiqon) with Maxima RNase H-minus RT (Thermo Fisher Scientific cat. no. EP0751) using a polyT primer containing the ISPCR sequence. Whole transcriptome amplification proceeded with KAPA HiFi HotStart ReadyMix using an ISPCR primer according to the following thermal program: 98C for 3 minutes, 21 cycles of 98C for 15 seconds, 67C for 20 seconds, and 72C for 6 minutes, and a final extension step of 72C for 5 minutes. The amplified cDNA was cleaned with 0.8X DNA SPRI beads (Beckman Coulter cat. no. B23318). Ten nanograms of amplified cDNA was tagmented at 58C for 10 minutes in a 10 uL reaction containing 2 uL of 5X tagmentation buffer (50 mM Tris-HCl, 25 mM MgCl2 pH 8.0), 2 uL of Tris Buffer (10 mM Tris-HCl, 1% Tween-20 pH 8), and 4 uL Nextera Tn5 transposase (Illumina). The reaction was stopped with 1% SDS and incubated at 72C for 10 minutes, then 4C for 3 minutes. The tagmented library was cleaned with 1X DNA SPRI beads followed by an indexing PCR with NEBNext High Fidelity polymerase to incorporate sample index barcodes and Illumina flow cell handles. The index PCR proceeded with the following thermal program: 72C for 3 minutes, 98C for 30 seconds, 12 cycles of 98C for 10 seconds, 60C for 30 seconds, 72C for 30 seconds, and a final extension step of 72C for 5 minutes. The final libraries were pooled, diluted and sequenced on a MiniSeq with a 75-cycle High Output Kit with the following read configuration: 36-8-8-38.

value: 1157.2 iPS cell line

value: iPSC-derived hematopoietic progenitor cells

value: UNC0224 1uM


title: F2-AsSug 5 uM 48h

value: Primary hepatocytes


title: KRAS_WT_day14_rep1_818

value: fibroblast cells

value: repRNA-v4 based


title: WT_LPS_01h_1

description:
IB13
BambuOutputcounts_gene.txt


title: F3-SugControl 48h


title: KRAS_WT_day1_rep1_779-1

value: No


title: WT_ctrl_2

description:
IB21
BambuOutputcounts_gene.txt


title: KRAS_WT_day1_rep2_779-2


title: F2-SugControl 48h


title: KRAS_S17N_day1_rep3_780-3


title: Whole_blood_HRFI_30_4325_0_13

description: castrated

value: neuter


title: Whole_blood_LRFI_24_4370_-5

description: castrated

value: LRFI


title: Whole_blood_LRFI_30_4381_2

description: castrated


title: Whole_blood_HRFI_30_4323_13

description: castrated

value: HRFI


title: F2-AsSug 50 uM 48h


title: RNA_T_UNC0224_1uM_1_rep2

description:
Bio rep 1, tech rep 2
T_UNC_1


title: Whole_blood_LRFI_24_4370_0_9

description: castrated


title: WT_ctrl_1

description:
GLKidneyWT1
BambuOutputcounts_gene.txt


title: F1-AsSug 10 uM 48h


title: RNA_T_UNC0224_1uM_2_rep2

description:
Bio rep 2, tech rep 2
T_UNC_2


title: KRAS_S17N_day1_rep1_780-1


title: F1-AsSug 5 uM 48h


title: F1-AsSug 50 uM 48h


title: RNA_T_UNC0224_1uM_1_rep1

description:
Bio rep 1, tech rep 1
T_UNC_1


title: F1-SugControl 48h

description: Hepatocyte isolated from individual fish were exposed to either arsenosugar (AsSug) at different concentrations (5, 10, 50 and 250 µM) in addition to control (AsSugControl) or arsenite (AsIII) at different concentrations (0.1, 1, 3 µM) in addition to control on individual plates for each compound.

value: AsSugControl


title: Whole_blood_HRFI_30_4323_1

description: castrated


title: KRAS_S17N_day1_rep2_780-2


title: Whole_blood_LRFI_30_4420_1

description: castrated


title: HTR8_Input_rep2

city: Shanghai

first: Wei

last: Li

address: 910# Hengshan Road, Shanghai, China.

institute: International Peace Maternity and Child Health Hospital

description:
RIP_H8-seq.count.txt
INPUT-B

data_processing:
PCR duplicates were marked with the Picard “Mark Duplicates” utility and removed from further analysis.
Gene annotated in NCBI were quantified using featureCounts
Peaks were called using MACS2 with the significance cut-off q-value <=0.01
Sequenced reads were trimmed for adaptor sequence and low-quality bases, then mapped to hg38 whole genome using Bowtie with standard alignment parameters.
Bam files were converted to BigWig files using deepTools.
Assembly: hg38
Supplementary files format and content: bigWig, MeRIP-seq peak (except for Input sample)
Supplementary files format and content: txt, counts for each RIP-seq sample

source_name: placenta

growth_protocol: The HTR-8/SVneo cells were cultured in DMEM/F12 (Gibco, Thermo Fisher Scientific-CN),medium was routinely supplemented with 10% fetal bovine serum (Hyclone, Thermo Fisher Scientific-CN) and 1% antibiotic solution (Gibco).

extract_protocol:
Total RNA from HTR-8/SVneo cells were extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instructions.
For the RNA-seq, the sequencing library was prepared using an Illumina mRNA-seq sample preparation kit and was then subjected to paired-end sequencing on the Illumina HiSeq platform.                                                      For the RIP-seq, the sequencing library was prepared deploying NEBNext Ultra Directional RNA Library Prep kit (New England Biolabs, USA) and sequenced on the Illumina HiSeq 3000 platform. For the MeRIP-seq, the library was prepared by smart-seq method. Both the input samples without IP and the m6A IP samples were subjected to 150-bp, paired-end sequencing on an Illumina NovaSeq 6000 sequencer.                                                                                For the ChIPseq, input amounts of DNA was converted into high quality libraries for next-generation sequencing on the Illumina platform using the NEBNext Ultra II DNA Library Prep Kit. Bead based size selection was performed for final library size selection to obtain ChIP-seq libraries containing fragments of 250 bp to 500 bp.

treatment_protocol: Before siRNA transfection, around 0.4 million HTR-8/SVneo cells  were seeded in to 6 well plate per

value: placenta

value: trophoblast cells


title: RNA_T_DMSO_2_rep2

description:
Bio rep 2, tech rep 2
T_DMSO_2

value: DMSO


title: KRAS_S17N_day14_rep3_823

value: molnupiravir


title: KRAS_S17N_day14_rep1_821


title: Whole_blood_HRFI_30_4447_0_18

description: castrated


title: Whole_blood_HRFI_30_3394_0_13

description: castrated


title: RNA_T_DMSO_2_rep1

description:
Bio rep 2, tech rep 1
T_DMSO_2


title: RNA_T_DMSO_1_rep1

description:
Bio rep 1, tech rep 1
T_DMSO_1


title: KRAS_S17N_day14_rep2_822


title: Whole_blood_LRFI_30_4307_2

description: castrated


title: RNA_T_DMSO_1_rep2

description:
Bio rep 1, tech rep 2
T_DMSO_1


title: HTR8_Input_rep1

description:
RIP_H8-seq.count.txt
INPUT-A


title: RNA_PBMC_T_2_rep1

description:
Bio rep 2, tech rep 1
PBMC_T_2

source_name: PBMCs

value: PBMCs

value: Peripheral blood T cells


title: StayGold_Post_1st_C7

description: no

data_processing:
Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: Coding sequence of EGFP or StayGold
Supplementary files format and content: The processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
Target genes (e.g., EGFP, StayGold) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing using Tsingke Biotechnology's Fast NGS service. In brief, the experimental workflow provided by Tsingke Biotechnology involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After another 5-7 days, a relatively stable and homogeneous population of polyclonal cells carrying replicative RNA with expression of EGFP or StayGold was generated (i.e., Parent). Approximately 1~2 million cells were passaged into a new 10-cm culture dish and treated with molnupiravir (10 µM) for two consecutive days with daily media changes. Then, they were divided equally into five separate 10-cm dishes and subjected to flow cytometry sorting after an additional cultivation period of approximately 3-4 days (i.e., Post_1st). After about another 7 days, these cells are expected to proliferate to a quantity of around 107. Similarly, these cells were exposed to a two-day treatment with molnupiravir (10 µM) and subsequently transferred onto five new 10-cm dishes for cultivation over a period of 3-4 days. The positive population was then sorted using flow cytometry at a ratio of 0.5%. The sorted cells were cultured to a count of approximately 106 and utilized for cryopreservation and total RNA isolation (i.e., Post_2nd).

value: kidney


title: HTR8_YTHDC1_rep2

description:
RIP_H8-seq.count.txt
YTH-B


title: RNA_PBMC_T_2_rep2

description:
Bio rep 2, tech rep 2
PBMC_T_2


title: Whole_blood_LRFI_30_4307_1

description: castrated


title: RNA_PBMC_T_1_rep2

description:
Bio rep 1, tech rep 2
PBMC_T_1


title: Whole_blood_LRFI_24_4381_-5

description: castrated


title: CS+ Gt time(dpi):5 rep:E [P63]

city: Harpenden

first: Daniel

last: Smith

state: Hertfordshire

address: West Common

department: IDE

country: United Kingdom

institute: Rothamsted Research

description: 150bp paired reads

data_processing:
HiSAT2 mapping using HPC EasyBuild v2.2.1-foss-2019b to IWGSC Chinese Spring v2.1
SAMtools format conversion, sorting & indexing using HPC Easybuild v1.10-GCC-9.3.0
Gene counts using HPC Easybuild SubRead featureCounts v2.0.0-GCC-8.3.0 and IWGSC annotation v43 for assembly v2.1, collated in file: takeAllCounts_wheat.tsv
Library normalisation and differential expression (DE) calling was carried out using the Bioconductor package DESeq2
Gene Ontology (GO) enrichment analysis was performed with web tools at GeneOntology (http://www.geneontology.org)using the Panther knowledgebase
Assembly: Chinese Spring Wheat IWGSC v2.1, annotation v43
Supplementary files format and content: takeAllCounts_wheat.tsv, featureCount gene counts

organism: Triticum aestivum

source_name: Chinese Spring & Gt-19d1

growth_protocol: Wheat seeds cv. Chinese Spring were surface sterilised with sodium hypochlorite and pre-germinated in a controlled environment growth chamber cabinet (20 °C day, 16 °C night, 16 hr light cycle) for two days. Three pre-germinated seeds were transplanted onto a square petri dish plate (12 cm x 12 cm) containing 1.5 % (w/v) water agar. Plates were placed horizontally in a growth cabinet (20 °C day, 16 °C night, 16 hr light cycle).

extract_protocol:
Following confocal assessment, 1 cm root samples at the same stage of fungal colonisation (harvested from the same plate) were pooled together to create a single sample for RNA extraction. RNA was extracted from frozen root material using the E.Z.N.A.® Plant RNA Kit with the associated RNase-free DNase I Set (Omega-Biotek, USA), following the standard protocol provided. RNA quality was assessed based on the RNA Integrity Number (RIN), measured using the Bioanalyser 2100 with the corresponding RNA 6000 Nano Kit (Agilent, USA), as per manufacturer instructions.
mRNA library preparation was carried out by Novogene (China) using the Novogene RNA Library Prep Set (PT042) for polyA enrichment.

treatment_protocol: After four days, one root from each plant was inoculated with a fungal plug (4 cm x 0.5 cm) cut from the leading edge of a fungal colony growing on 1.5 % water agar . Five replicates were prepared for each treatment. Roots were sampled daily from 2-6 dpi. Briefly, two 1 cm root samples were harvested from the inoculated area and snap frozen in liquid nitrogen for RNA extraction

value: root

tag: cultivar

value: var. Chinese Spring

value: CS+ Gt time(dpi):5 rep:E


title: StayGold_Post_2nd_C13

description: no

value: fibroblast cells


title: StayGold_parent_C1

description: no

value: repRNA-v4 based


title: RNA_PBMC_T_1_rep1

description:
Bio rep 1, tech rep 1
PBMC_T_1


title: HTR8_YTHDC1_rep1

description:
RIP_H8-seq.count.txt
YTH-A


title: CS+ Gt time(dpi):5 rep:D [P50]

description: 150bp paired reads

value: CS+ Gt time(dpi):5 rep:D


title: Whole_blood_LRFI_30_4381_13

description: castrated

value: neuter


title: EGFP_Post_2nd_P2_rep2_664

description: no


title: EGFP_Post_2nd_P2_rep3_664

description: no


title: RNA_EHT_UNC0224_500nM_2_rep2

description:
Bio rep 2, tech rep 2
EHT_UNC_Low_2

value: 1157.2 iPS cell line

value: iPSC-derived hematopoietic progenitor cells


title: Whole_blood_HRFI_24_3386_0_9

description: castrated

value: HRFI


title: CS+ Gt time(dpi):5 rep:C [P34]

description: 150bp paired reads

value: CS+ Gt time(dpi):5 rep:C


title: RNA_EHT_UNC0224_500nM_1_rep2

description:
Bio rep 1, tech rep 2
EHT_UNC_Low_1


title: EGFP_Post_2nd_P1_rep3_624

description: no


title: CS+ Gt time(dpi):5 rep:A [P2]

description: 150bp paired reads

value: CS+ Gt time(dpi):5 rep:A


title: EGFP_Post_2nd_P1_rep2_624

description: no


title: Whole_blood_HRFI_24_4462_-5

description: castrated


title: Whole_blood_HRFI_30_3394_0_18

description: castrated


title: Whole_blood_LRFI_30_3357_2

description: castrated

value: LRFI


title: RNA_EHT_UNC0224_500nM_1_rep1

description:
Bio rep 1, tech rep 1
EHT_UNC_Low_1


title: CS+ Gt time(dpi):4 rep:E [P74]

description: 150bp paired reads

value: CS+ Gt time(dpi):4 rep:E


title: RNA_EHT_UNC0224_1uM_2_rep2

description:
Bio rep 2, tech rep 2
EHT_UNC_High_2

value: UNC0224 1uM


title: EGFP_Post_2nd_P1_rep1_624

description: no

value: molnupiravir


title: Whole_blood_HRFI_30_4323_2

description: castrated


title: Whole_blood_LRFI_30_4420_0_13

description: castrated


title: EGFP_Post_1st_rep2_571

description: no

value: kidney


title: CS+ Gt time(dpi):4 rep:D [P46]

description: 150bp paired reads

value: root

value: CS+ Gt time(dpi):4 rep:D


title: EGFP_Post_2nd_P2_rep1_664

description: no


title: RNA_EHT_UNC0224_500nM_2_rep1

description:
Bio rep 2, tech rep 1
EHT_UNC_Low_2


title: Whole_blood_HRFI_24_4325_0_9

description: castrated


title: Whole_blood_LRFI_30_4381_0_18

description: castrated


title: RNA_EHT_UNC0224_1uM_1_rep2

description:
Bio rep 1, tech rep 2
EHT_UNC_High_1


title: EGFP_Parent_rep3_500

description: no

value: No


title: CS+ Gt time(dpi):4 rep:B [P16]

description: 150bp paired reads

value: var. Chinese Spring

value: CS+ Gt time(dpi):4 rep:B


title: CS+ Gt time(dpi):5 rep:B [P27]

description: 150bp paired reads

value: CS+ Gt time(dpi):5 rep:B


title: Whole_blood_HRFI_30_3386_2

description: castrated

value: neuter


title: CS+ Gt time(dpi):4 rep:A [P3]

description: 150bp paired reads

value: CS+ Gt time(dpi):4 rep:A


title: RNA_EHT_DMSO_2_rep2

description:
Bio rep 2, tech rep 2
EHT_DMSO_2

value: 1157.2 iPS cell line

value: iPSC-derived hematopoietic progenitor cells

value: DMSO


title: EGFP_Parent_rep2_500

description: no

value: fibroblast cells

value: repRNA-v4 based


title: Whole_blood_HRFI_30_3394_2

description: castrated

value: HRFI


title: CS+ Gt time(dpi):2 rep:E [P73]

description: 150bp paired reads

value: CS+ Gt time(dpi):2 rep:E


title: EGFP_Parent_rep1_500

description: no


title: RNA_EHT_UNC0224_1uM_2_rep1

description:
Bio rep 2, tech rep 1
EHT_UNC_High_2

value: UNC0224 1uM


title: MEK1_NGS_Day7_261

data_processing:
The resulting Pacbio sequencing data were demultiplexed based on samples and barcodes using in-house Python scripts. The MEK1 region was aligned to MEK1 reference sequence using BWA 0.7.17-r1188. Mutation (mismatch) information for each sequencing read was obtained from the bam file using pysam 0.18.0 and custom Python scripts. Besides, to analyze the NGS data, Trimmomatic 0.39 was used to remove the 5' end 15-bp low quality sequence of the FASTQ file. BWA 0.7.17-r1188 was employed to map the resulting cleaned reads to the reference sequence. The resulting bam files were processed using a command mpileup from samtools to generate bcf file. To precisely profile the mutational frequency, nucleotides with sequencing quality score below Q30 were discarded. Then, custom Bash and Python scripts were used to extract the mutation information and calculate the mutation frequency. The mutation frequency at each specific position within the target gene is calculated by dividing the number of mutated bases (transversion and transition) at that position by the total number of bases sequenced at the specific position.
Assembly: Coding sequence of MEK1 from H. sapiens
Supplementary files format and content: The processed files in txt format contain information about the sequenced read number for each barcode and the mutation (mismatch) number for each read. While the processed files in xlsx format contain information such as the number of aligned bases per position, the number of base mutations per mutation type (i.e., A>T, A>G, A>C…), the count of base deletions and insertions, the total number of mapped bases, and the frequency of base mutations.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
For Pacbio sequencing, The MEK1-T2A-PuroR barcode region was amplified from both the DNA library plasmid and the cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition. For Fast NGS sequencing, Target gene (e.g., MEK1) were amplified from the cDNA samples via 28 rounds of PCR using KOD OneTM PCR Master Mix and primers specific to each target (For a 50 μl PCR reaction, 5 μl cDNA was used as template). The PCR product was purified using the HiPure Gel Pure DNA Micro Kit (Magen, #D2110-03). The gel-purified PCR product (approximately 1 μg) was subjected to sequencing. In brief, the experimental workflow involved the following steps: Firstly, DNA was enzymatically digested using Tn5 with sequence adaptors. Secondly, the digested products were subjected to PCR amplification using indexed sequencing primers. Finally, the resulting PCR products were recovered and subjected to sequencing using an Illumina MiSeq platform, ensuring a sequencing depth exceeding 500× on the target gene for accurate estimation of mutation frequency.

treatment_protocol: Medium was replaced with fresh medium containing 5 μg/mL puromycin 24 h post-electroporation (i.e., Day 1). After 4 days of selection by puromycin (i.e., on Day 5), approximately 2 million of cells were transferred to a new 10-cm dish and subjected to RNA mutagenesis using a concentration of 2 μM molnupiravir. On Day 7,therapeutic drug (2-5 μM cobimetinib) was added into the medium for selection. Puromycin and molnupiravir were also added to the medium simultaneously. One week later (i.e., Day 14), survived cells were collected for total RNA extraction, sequencing, and mutation analysis.

value: kidney

value: molnupiravir


title: RNA_EHT_DMSO_2_rep1

description:
Bio rep 2, tech rep 1
EHT_DMSO_2


title: CS+ Gt time(dpi):2 rep:D [P58]

description: 150bp paired reads

value: root

value: CS+ Gt time(dpi):2 rep:D


title: CS+ Gt time(dpi):4 rep:C [P31]

description: 150bp paired reads

value: var. Chinese Spring

value: CS+ Gt time(dpi):4 rep:C


title: CS+ Gt time(dpi):2 rep:C [P37]

description: 150bp paired reads

value: CS+ Gt time(dpi):2 rep:C


title: RNA_EHT_DMSO_1_rep2

description:
Bio rep 1, tech rep 2
EHT_DMSO_1


title: MEK1_NGS_Day5_257


title: EGFP_Post_1st_rep1_571

description: no


title: RNA_EHT_DMSO_1_rep1

description:
Bio rep 1, tech rep 1
EHT_DMSO_1


title: RNA_EHT_UNC0224_1uM_1_rep1

description:
Bio rep 1, tech rep 1
EHT_UNC_High_1


title: Whole_blood_HRFI_24_4323_-5

description: castrated

value: neuter


title: MEK1_NGS_Day14_270


title: MEK1_NGS_Day1_252


title: Whole_blood_LRFI_30_4420_13

description: castrated

value: LRFI


title: CS+ Gt time(dpi):2 rep:B [P19]

description: 150bp paired reads

value: CS+ Gt time(dpi):2 rep:B


title: Whole_blood_HRFI_24_4325_-5

description: castrated


title: Tancarville 2500 mg/L male biol rep 3

city: Le Havre

first: Caroline

last: Arcanjo

address: 25, rue Philippe Lebon

institute: UMR-I02 SEBIO

data_processing:
Raw sequencing reads are quality trimmed and filtered for average quality. Reads containing any uncalled bases denominated with an ‘N’ are also removed. Any PhiX reads that happen to have bled into any of the demultiplexed samples are also discarded
The surviving sequencing reads were then mapped to the GCF_000591075.1 (Eurytemora_affinis_Eaff_2.0) reference sequence downloaded from NCBI on 25.02.2021
The STAR splice aware RNA mapping software version 2.7.7a was used to create the reference mapping index using the reference genome and map the surviving sequencing reads using default parameters.
The Salmon RNA quantification software version 1.4.0 was used to map and quantify the surviving sequencing reads against the reference transcriptome using default parameters
The reads that mapped uniquely to reference-annotated genes were counted using the HTSeq-count software version 0.11.2.
The read counts were normalized using the method implemented in the DESeq2 R package
Assembly: GCF_000591075.1 (Eurytemora_affinis_Eaff_2.0)
Supplementary files format and content: Tab-delimited text files with gene counts for each samples

organism: Eurytemora affinis

source_name: entire adults

growth_protocol: Copepods were sampled in the Seine estuary. The transfer to the laboratory was performed into cool boxes containing Seine water. Salinity was gradually adjusted to 15 psu (practical salinity unit), using a mixture of UV-treated filtered seawater and dechlorinated tap water in the laboratory. Copepods were then collected through 355 µm mesh size sieves to remove nauplii and copepodites and transferred to a clean 15 psu water glass tank. They were fed ad libitum with Rhodomonas salina algae and kept under optimal conditions (i.e., 15 °C, 15 psu, 12 h/12 h light-dark cycle, Devreker et al 2009) for at least three days for acclimatization.

extract_protocol:
Total RNA extraction was performed using the Quick-RNA™ Tissue/Insect Microprep (Zymo Research, Irvine, CA, USA) following manufacturer instructions.
Sequencing libraries were performed using the Universal Plus mRNA Seq Library Preparation with NuQuant (Nugen Tecan Genomics) from 200 ng of total RNA and were qualified on High Sensitivity DNA chip on 2100 Bioanalyzer from Agilent Technologies.

treatment_protocol: Copepod were exposed to sediment-derived elutriats at two concentration (2500 mg/L and 350 mg/L) for both sites. Control groups were only exposed to water.

value: entire adults

tag: Sex

value: male

tag: Stage

value: adult

tag: exposure time

value: Tancarville 2500 mg/L


title: CS + Gh time(dpi):5 rep:E [P67]

description: 150bp paired reads

source_name: Chinese Spring & Gh-2C17

tag: tissue

value: CS + Gh time(dpi):5 rep:E


title: CS+ Gt time(dpi):2 rep:A [P4]

description: 150bp paired reads

value: CS+ Gt time(dpi):2 rep:A


title: Tancarville 2500 mg/L male biol rep 2


title: Tancarville 2500 mg/L male biol rep 1


title: Whole_blood_LRFI_30_3371_1

description: castrated


title: CS + Gh time(dpi):5 rep:D [P48]

description: 150bp paired reads

value: CS + Gh time(dpi):5 rep:D


title: Whole_blood_HRFI_30_3386_0_18

description: castrated

value: HRFI


title: Whole_blood_LRFI_30_4307_0_13

description: castrated


title: SCAF2349_V56

city: Bethesda

first: Kimia

last: Dadkhah

address: Rockville Pike

department: NCI

institute: NIH

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v6.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: Enhanced_Macaca_mulatta_10.104
Supplementary files format and content: h5 files: H5 is a binary format that can compress and access data much more efficiently than text formats such as MEX, which is especially useful when dealing with large datasets.

organism: Macaca mulatta

source_name: PBMC

growth_protocol: PBMC isolated from fresh blood

extract_protocol:
The samples were washed twice with PBS
Library construction was performed according to the manufacter’s instructions (single cell 3’ v3.1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.

treatment_protocol: No treatment

value: PBMC

tag: condition

value: vaccinated


title: Tancarville 350 mg/L male biol rep 3

value: Tancarville 350 mg/L


title: Whole_blood_LRFI_30_4370_0_13

description: castrated


title: MEK1_Pacbio_dna

value: kidney

tag: cell line

tag: cell type

value: fibroblast cells

value: repRNA-v4 based

value: No


title: MEK1_Pacbio_day7

tag: treatment

value: molnupiravir


title: CS + Gh time(dpi):5 rep:B [P30]

description: 150bp paired reads

value: root

value: var. Chinese Spring

value: CS + Gh time(dpi):5 rep:B


title: Tancarville 350 mg/L male biol rep 1

value: entire adults

value: male

value: adult


title: Whole_blood_HRFI_30_4462_2

description: castrated

value: neuter


title: Fatouville 2500 mg/L male biol rep 3

value: Fatouville 2500 mg/L


title: MEK1_Pacbio_day5

tag: genotype


title: Whole_blood_HRFI_24_4447_0_9

description: castrated

value: HRFI


title: CS + Gh time(dpi):4 rep:E [P61]

description: 150bp paired reads

value: CS + Gh time(dpi):4 rep:E


title: CS + Gh time(dpi):5 rep:A [P9]

description: 150bp paired reads

value: CS + Gh time(dpi):5 rep:A


title: SCAF2350_N2

description: 10x Genomics

value: PBMC

value: naïve


title: Whole_blood_LRFI_30_4381_1

description: castrated

value: LRFI


title: Whole_blood_LRFI_30_3357_1

description: castrated


title: Whole_blood_LRFI_30_3371_13

description: castrated


title: MEK1_Pacbio_day1

value: kidney

value: fibroblast cells

value: repRNA-v4 based


title: Fatouville 2500 mg/L male biol rep 2


title: CS + Gh time(dpi):4 rep:D [P57]

description: 150bp paired reads

value: root

value: CS + Gh time(dpi):4 rep:D


title: Fatouville 350 mg/L male biol rep 3

value: Fatouville 350 mg/L


title: Tancarville 350 mg/L male biol rep 2

value: entire adults

value: Tancarville 350 mg/L


title: CS + Gh time(dpi):4 rep:C [P43]

description: 150bp paired reads

value: var. Chinese Spring

value: CS + Gh time(dpi):4 rep:C


title: CS + Gh time(dpi):5 rep:C [P44]

description: 150bp paired reads

value: CS + Gh time(dpi):5 rep:C


title: Fatouville 350 mg/L male biol rep 2

value: male

value: adult


title: Whole_blood_LRFI_30_4370_2

description: castrated

value: neuter


title: kras_s17n_dna_barcode_CCCAGACGGCTCGGCATAGC

data_processing:
The resulting Pacbio sequencing data were demultiplexed based on samples and barcodes using in-house Python scripts. The KRAS (S17N) region was aligned to KRAS (S17N) reference sequence using BWA 0.7.17-r1188. Mutation (mismatch) information for each sequencing read was obtained from the bam file using pysam 0.18.0 and custom Python scripts.
Assembly: CDS of KRAS (S17N) from H. sapiens
Supplementary files format and content: The processed files in txt format contain mutation (mismatch) information for each read.

extract_protocol:
The cells collected during the directed evolution experiments were harvested, and total RNA was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, #15596018). Subsequently, approximately 3 μg of total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-02) with a primer (SINDBIS_GSPR2: TGCGCCTCCTCGGAAATACG) located in the 3′ untranslated region of the replicative RNA.
The KRAS (S17N)-T2A-PuroR barcode region was amplified from both the DNA library plasmid and the cDNA libraries using indexed primers. The purified PCR products were mixed and subjected to adapter ligation, fragment selection, primer and polymerase binding, followed by sequencing using a PacBio Sequel IIe instrument for Circular Consensus Sequencing (CCS) reads acquisition.

value: no


title: Fatouville 2500 mg/L male biol rep 1

value: Fatouville 2500 mg/L


title: Fatouville 350 mg/L male biol rep 1


title: kras_s17n_day9_barcode_CCCAGACGGCTCGGCATAGC

value: molnupiravir


title: MEK1_Pacbio_day14

value: kidney


title: kras_s17n_day1_barcode_CCCAGACGGCTCGGCATAGC

value: fibroblast cells


title: kras_s17n_day5_barcode_CCCAGACGGCTCGGCATAGC

value: repRNA-v4 based


title: Whole_blood_LRFI_30_3357_0_13

description: castrated

value: LRFI


title: CS + Gh time(dpi):4 rep:B [P23]

description: 150bp paired reads

value: root

value: CS + Gh time(dpi):4 rep:B


title: Whole_blood_HRFI_24_3386_-5

description: castrated

value: HRFI


title: CS + Gh time(dpi):2 rep:E [P69]

description: 150bp paired reads

value: CS + Gh time(dpi):2 rep:E


title: Control male biol rep 3

value: entire adults

value: Control


title: CS + Gh time(dpi):2 rep:C [P39]

description: 150bp paired reads

value: var. Chinese Spring

value: CS + Gh time(dpi):2 rep:C


title: Whole_blood_LRFI_30_3371_0_18

description: castrated


title: CS + Gh time(dpi):2 rep:D [P47]

description: 150bp paired reads

value: CS + Gh time(dpi):2 rep:D


title: Whole_blood_HRFI_30_4462_1

description: castrated

value: neuter


title: Whole_blood_HRFI_24_4323_0_9

description: castrated


title: kras_s17n_day14_barcode_CCCAGACGGCTCGGCATAGC


title: Control male biol rep 2

value: male

value: adult


title: Whole_blood_HRFI_30_4447_0_13

description: castrated


title: CS + Gh time(dpi):4 rep:A [P11]

description: 150bp paired reads

value: CS + Gh time(dpi):4 rep:A


title: CS + Gh time(dpi):2 rep:B [P25]

description: 150bp paired reads

value: CS + Gh time(dpi):2 rep:B


title: Whole_blood_HRFI_30_4325_1

description: castrated


title: kras_s17n_dna_barcode_GCTATAAGCCCCCTACAAAG

value: kidney

value: fibroblast cells

value: no


title: CS + Gh time(dpi):2 rep:A [P13]

description: 150bp paired reads

value: root

value: CS + Gh time(dpi):2 rep:A


title: Whole_blood_HRFI_30_4323_0_13

description: castrated

value: HRFI


title: Anti-NY-ESO-1- transduced T-cells (Donor 20)

city: Novosibirsk

first: Roman

middle: Yurievich

last: Perik-Zavodskii

state: Novosibirsk region

address: Yadrincevskaya 14

country: Russia

institute: "Research Institute of Fundamental and Clinical Immunology" (RIFCI)

description: We performed mRNA profiling for 579 target genes, for 14 housekeeping genes, for 8 negative and 6 positive spike-in controls

hyb_protocol: 100ng of total RNA was hybridized with NanoString Immunology V2 panel according to the manufacturer's protocols.

scan_protocol: not provided

data_processing: We performed QC on the raw data, normalized gene expression using 15 housekeeping genes, removed the non-expressing genes that had counts below the sum(mean of negative controls + 2 SD + mean of POS_E controls) in every studied sample.

label: not provided

source_name: Anti-NY-ESO-1- transduced T-cells

label_protocol: not provided

extract_protocol: We obtained peripheral blood mononucler cells using Ficoll-paque density gradient, co-cultured them with anti-CD3 beads, transduced the resulting T-cells with the MMLV-based retrovirus containing the anti-NY-ESO-1 construct.

value: 23 years

value: female

value: Peripheral blood

value: Anti-NY-ESO-1- transduced T-cells


title: kras_s17n_dna_barcode_AGACATTGCCATAAACGAAG

value: repRNA-v4 based


title: kras_s17n_day9_barcode_AGACATTGCCATAAACGAAG

value: molnupiravir


title: Anti-NY-ESO-1- transduced T-cells (Donor 19)


title: kras_s17n_day9_barcode_GCTATAAGCCCCCTACAAAG


title: kras_s17n_day5_barcode_GCTATAAGCCCCCTACAAAG


title: CS + Control time(dpi):5 rep:E [P65]

description: 150bp paired reads

source_name: Chinese Spring

value: var. Chinese Spring


title: CS + Control time(dpi):5 rep:D [P49]

description: 150bp paired reads


title: Anti-NY-ESO-1- transduced T-cells (Donor 18)


title: Control male biol rep 1

value: entire adults

value: male

value: Control


title: CS + Control time(dpi):5 rep:C [P35]

description: 150bp paired reads


title: CS + Control time(dpi):5 rep:B [P21]

description: 150bp paired reads


title: CS + Control time(dpi):5 rep:A [P14]

description: 150bp paired reads


title: Anti-NY-ESO-1- transduced T-cells (Donor 17)

value: 24 years


title: kras_s17n_day1_barcode_AGACATTGCCATAAACGAAG

value: kidney


title: kras_s17n_day14_barcode_GCTATAAGCCCCCTACAAAG

value: fibroblast cells


title: Anti-NY-ESO-1- transduced T-cells (Donor 16)

value: 26 years


title: kras_s17n_day14_barcode_AGACATTGCCATAAACGAAG


title: Whole_blood_LRFI_30_4420_0_18

description: castrated

value: LRFI

value: neuter


title: Anti-NY-ESO-1- transduced T-cells (Donor 15)

value: 25 years


title: Whole_blood_LRFI_24_4381_0_9

description: castrated


title: CD3+ T-cells (Donor 20)

source_name: CD3+ T-cells

value: 23 years

value: CD3+ T-cells


title: CS + Control time(dpi):4 rep:D [P51]

description: 150bp paired reads

value: root


title: Whole_blood_LRFI_30_3371_2

description: castrated


title: Whole_blood_HRFI_30_4462_0_18

description: castrated

value: HRFI


title: CS + Control time(dpi):4 rep:C [P38]

description: 150bp paired reads


title: Whole_blood_HRFI_30_4447_13

description: castrated


title: Whole_blood_HRFI_30_3394_13

description: castrated


title: CD3+ T-cells (Donor 18)

value: female

value: Peripheral blood


title: CD3+ T-cells (Donor 17)


title: Whole_blood_LRFI_24_4307_-5

description: castrated


title: CS + Control time(dpi):4 rep:B [P17]

description: 150bp paired reads

value: var. Chinese Spring


title: PY63024_03

city: Houston

first: Matthew

last: Dysthe

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

hyb_protocol: none provided

scan_protocol: none provided

data_processing:
The raw data was analyzed with the nSolver Analysis Software 3.0 program to generate the normalized data. The median values of negative controls were used for background subtraction and the geometric mean was used for normalization.
Each processed sample is normalized counts generated by nSolver Analysis Software 3.0 program.

extract_protocol: Qiagen RNEasy Micro Kit

value: Primary Human NK Cells

tag: donor

tag: cytokine condition

tag: day


title: CS + Control time(dpi):4 rep:E [P68]

description: 150bp paired reads


title: PY63022_01


title: CD3+ T-cells (Donor 16)

tag: age


title: PY63026_05


title: PY63025_04


title: PY63027_06


title: CD3+ T-cells (Donor 19)


title: CS + Control time(dpi):2 rep:E [P64]

description: 150bp paired reads


title: CD3+ T-cells (Donor 15)

value: male


title: PY63023_02


title: CS + Control time(dpi):2 rep:D [P54]

description: 150bp paired reads


title: Whole_blood_HRFI_30_3394_1

description: castrated

tag: repetition

tag: breed

tag: pen


title: KO2

city: St. Louis

first: Zhiyu

last: Dai

state: MO

address: 4960 Childrens Place

institute: Washington University in St. Louis

description:
0027365_4_c
fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression
mRNA in situ

hyb_protocol: Fluorescent in situ hybridization (FISH) and imaging–based.

scan_protocol: Cell segmentation was based on tissue using a combination of nucleus (DAPI), cellular content, and cell boundary markers with the Xenium Multi-Tissue Stain Mix (10X Genomics, PN 2000991). Cell and tissue landmarks are further delineated through HE staining.

data_processing:
Xenium data out was loaded on Xenium Explorer for general data visualization and analysis. For unsupervised clustering of the dataset, Xenium data out was processed using Scappy package and Seurat packages. For deconvolution, the LungMap Mouse Reference v.1.1 dataset was used for annotation. Single Cell Proportion Test was performed using scProportionTest. The DEGs visualization was generated using scRNAtoolVis.
Differential gene expression analyses between WT and KO samples for each cluster of interest were performed using the Wilcoxon Rank Sum Test algorithm with log normalized counts in the RNA assay as input. A threshold of 0.25 for log fold change, 0.05 for the adjusted P value and 0.1 for minimal fraction of cells was applied for downstream analysis. A permutation test is used to calculate a p-value for each cluster, and a confidence interval for the magnitude difference is returned via bootstrapping. FDR<0.05 was designated as significant.

source_name: fixed frozen mouse lung section

label_protocol: The predesigned 379-gene mouse panel of probes provided by 10x Genomics was used. We conducted further analysis using Seurat3, Scanpy4 and Squidpy5 packages. Each tissue section was provided as a separate dataset, with cell segmentation already implemented on the Xenium analyzer.

growth_protocol: Egln1f/f (WT) and Egln1Tie2Cre (KO) mice were generated as described previously (Zhiyu Dai, et al. Circulation). Mice at the age of 2 to 3.5 months were used in the study.

extract_protocol: Mouse lung was first fixed by 4% PFA through transcardial perfusion, dissected out, and further post-fixed in 4% PFA overnight at 4 °C. The lung was then cryoprotected, embedded in OCT compound, and sectioned on a cryostat into 10um thickness. And then mounted on the Xenium slide imaging area.

value: Egln1Tie2Cre (KO)

value: fixed frozen mouse lung section


title: Whole_blood_LRFI_30_4420_2

description: castrated

tag: temperature

tag: gender


title: KO1

description:
0027365_3_d
fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression
mRNA in situ

label: predesigned 379-gene mouse panel of probes


title: WT2

description:
0027365_2_e
fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression
mRNA in situ

value: littermate Egln1f/f (WT) control


title: pC3_CLSY3_3xF_BLRP_smRNAseq_flowerbuds

city: Strasbourg

first: Todd

middle: L

last: Blevins

address: 12 Rue du Général-Zimmer

institute: CNRS

data_processing:
Illumina Casava software used for basecalling and demultiplexing.
All samples were processed as follows. Raw reads were trimmed using cutadapt (v1.18) to remove adapters (-a AACTGTAGGCACCATCAAT) and reads shorter than 15 nt (-m 15). Trimmed reads were mapped to the Arabidopsis TAIR10 genome using ShortStack (v3.8.5) allowing one mismatch (--mismatches 1) and allowing multi-mapped reads that will be guided to a specific location with the fractional-seeded algorithm (-mmap f). To meet the characteristics of Pol IV transcription, the mapped reads were further filtered to keep only perfectly-mapped reads or reads with a single mismatch at the 3’-ends using the filter function of bamtools (v2.5.1) together with a previously published script JSON_findPerfectMatches_and_TerminalMisMatches_v3. To facilitate small RNA quantification, Tag Directories were generated from the filtered bam files using the makeTagDirectory function of HOMER (v4.10) with the following options: -format sam -mis 1 -keepAll. Split Tag Directories with a certain smRNA size (21-24 nt) were made using a previously published perl script splitTagDirectoryByLength.dev2.pl.
Assembly: TAIR10
Supplementary files format and content: bedGraph file, including header and chr#, start position, end position and normalized value (rp10M).

organism: Arabidopsis thaliana

source_name: stage 12 and younger flower buds

growth_protocol: Growth conditions and tissue collection for FB for all genotypes were as follows: seeds were sterilized with ethanol and plated on half-strength Linsmaier and Skoog medium (1/2 LS). After three days of stratification in the dark at 4℃, plants were grown in the Percival growth chamber (GCU-36L5) under 16h-light and 8h-dark cycle at 22℃ for 7 days and then were transplanted onto the soil in Salk’s greenhouse with long-day conditions. The first inflorescence of 6 plants was collected in a tube, immediately frozen in liquid nitrogen, and kept at -80°C until use.

extract_protocol:
Total RNAs were isolated using Trizol reagent (Life Tech, Cat# 15596018) following the manual and dissolved in 250μL RNase-free water. 10.0 µg of total RNA from FB were used for smRNA enrichment through a polyethylene glycol (PEG) precipitation step. One volume of a 20% PEG 8000/2M NaCl solution was added to each RNA sample, mixed well, kept on ice for more than 30 minutes (min), and then centrifuged at 13,000rpm for 15 min at 4°C. The supernatant, containing the small RNAs, was collected and precipitated with 1 volume of cold isopropanol, mixed, and incubated on ice for 30 min. Then, the small RNA samples were recovered by centrifuging at 13,000rpm for 30 min at 4°C. The resulting small RNA samples were run on 15% polyacrylamide-8M urea gels and performed size-selection of fragments from 17-29 nucleotide (nt) following the ZR-small RNA ladder (Zymo Research, Cat# R1090). The small RNAs in the gels were recovered by incubating with 0.3M NaCl overnight, filtering the gels, and adding 2.5 volumes of 100% ethanol and 3μL of glycogen for precipitation over 2 hours (h) at -80°C.
The small RNAs were used for library preparation with the NEBnext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Cat# E7300) following the user’s manual. The final library products were further purified using an 8% polyacrylamide gel to excise 130-160nt products relative to the pBR322 DNA-MspI Digest ladder (New England Biolabs, Cat# E7323AA). The purified libraries were pooled and sequenced (single end 50 bp, SE50) on a NextSeq 2000 machine.

value: stage 12 and younger flower buds

value: pC3_CLSY3_3xF_BLRP clsy3_1


title: clsy3_2023_smRNAseq_flowerbuds


title: WT_rep2_smRNAseq_flowerbuds

value: wild-type


title: CS + Control time(dpi):2 rep:C [P42]

description: 150bp paired reads

tag: cultivar


title: WT1

description:
0027365_1_f
fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression
mRNA in situ


title: CS + Control time(dpi):2 rep:B [P26]

description: 150bp paired reads


title: WT_rep3_smRNAseq_flowerbuds


title: Whole_blood_LRFI_30_4370_0_18

description: castrated

value: LRFI

value: neuter


title: CS + Control time(dpi):2 rep:A [P5]

description: 150bp paired reads


title: WT_rep1_smRNAseq_flowerbuds


title: ctrl dataset II

status: Public on Sep 29 2024

city: Philadelphia

first: Priyankara

middle: J

last: Wickramasinghe

state: PA

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

description:
ctrl
control

data_processing:
Preprocessing of scRNA-seq data was done using CellRanger Suite (v3.1.0, https://support.10xgenomics.com) with refdata-cellranger-mm10-3.0.0 transcriptome as a reference
Assembly: mm10
Supplementary files format and content: standard cellranger output

source_name: lymph node

extract_protocol:
Single cell droplets were generated using the Chromium next gem single cell 3' kit v3.1 (10x Genomics)
cDNA synthesis and amplification, library preparation and indexing were done using the 10x Genomics Library Preparation kit (10x Genomics)
10x genomics single cell RNA-seq

value: lymph node

value: ctrl


title: 2DG_aPDL1 dataset II

description:
2DG_aPDL1
Treatment, 2-Deoxy-D-glucose (2DG) _ aPDL1


title: Whole_blood_HRFI_30_4325_0_18

description: castrated


title: 2DG dataset II

description:
2DG
Treatment, 2-Deoxy-D-glucose (2DG)


title: aPDL1 dataset II

description:
aPDL1
Treatment, aPDL1


title: MB4132-rna

first: Marco

last: Gallo

address: 1102 Bates Avenue

department: Pediatrics

data_processing:
Fastqs were processed to hic files using Juicer v1.6.
Contact domains were annotated from hic files using Arrowhead from Juicer Tools v1.19.02 with parameters ‘--ignore-sparsity -k SCALE’ for the following data resolutions (kb): 10, 25, 50, 100.
Loops were annotated from hic files using HiCCUPS from Juicer Tools v1.19.02 with parameters ‘--cpu -m 4096 --ignore-sparsity -k SCALE’ for resolutions (kb): 10, 25.
Compartments were annotated from hic files using the leading eigenvectors of the Juicer Tools Pearsons with parameters ‘SCALE BP 50000 -p’
Boundary scores were annotated from 50-kb contact matrices using Robustad v1.0
RNA-seq alignment: reads aligned to hg38 (GCA_000001405.15_GRCh38_no_alt_plus_hs38d1_analysis_set) using HISAT2 (v2.1.0) with parameters ‘--rna-strandness RF --downstream-transcriptome-assembly’
RNA-Seq quantification: SAM output was sorted, converted to BAM, and indexed using samtools (v1.9). A counts table was prepared using the count function of HTSeq (v0.13).
RNA-Seq differential expression testing: Limma with Voom transformation (v3.44)
RNA-Seq coverage: BigWig tracks were generated using DeepTools bamCoverage with parameters --effectiveGenomeSize 2913022398 --normalizeUsing RPGC --binSize 1. Separate files for positive and negative strand were generated with parameters --filterRNAstrand "forward" or  --filterRNAstrand "reverse"
Assembly: hg38, GCA_000001405.15_GRCh38_no_alt_plus_hs38d1_analysis_set
Supplementary files format and content: TSV; BoundaryScores.max.50kb.tsv: Summary table of RobusTAD scores for all samples
Supplementary files format and content: BDG; BoundaryScores.max.50kb.bedgraph: BEDGRAPH format values from RobusTAD boundary quantification
Supplementary files format and content: WIG; BoundaryScores.max.50kb.wig: WIGGLE format values from RobusTAD boundary quantification
Supplementary files format and content: TSV; TADBoundaryCalls.50kb.tsv: Summary table of RobusTAD boundaries for all samples
Supplementary files format and content: WIG; eigenvalue_allChr_50kb.wig: Eigenvalues of leading PC indicating genome compartmentalization
Supplementary files format and content: TSV; compartment_eigenvalues.tsv: Summary table of eigenvalues for all samples
Supplementary files format and content: HIC; inter_30.hic: Hi-C contact maps generated by Juicer
Supplementary files format and content: BigWig; pos.bw: RNA-seq coverage corresponding to the positive strand
Supplementary files format and content: BigWig; neg.bw: RNA-seq coverage corresponding to the negative strand
Supplementary files format and content: TSV; limma.*.wrt.*.tsv: Differential expression calls between tumor subgroups generated using limma
Supplementary files format and content: TSV; union_arrowhead_scores.tsv: Summary table of Arrowhead contact domain scores for all samples
Supplementary files format and content: BEDPE; merged_loops.bedpe: Loop calls generated using HiCCUPS
Supplementary files format and content: TSV; union_hiccups_scores.tsv: Summary table of HiCCUPS loop scores for each sample

source_name: brain

growth_protocol: The majority of samples were uncultured, primary patient tissue. The brain tissue culture was grown as described previously (Michealraj et al. Cell. 2020; PMID: 32445698).

extract_protocol:
Approximately 2.5M cells were used for In-situ Hi-C library approach (Rao et al. Cell. 2014; PMID: 25497547) with library preparation performed as described previously (Johnston et al. Genome Research. PMID: 31249064). Hi-C libraries were sequenced at 150 bp PE with a Hi-Seq X instrument (Illumina) at McGill Genome Centre (Montreal, QC).
In-situ Hi-C
Kapa RNA HyperPrep kit prepared and sequenced at Ontario Institute for Cancer Research (OICR)
Paired-end, stranded RNA-Seq

value: brain

tag: Sex

value: M

tag: disease

value: medulloblastoma

tag: disease subgroup


title: MB4174-rna

tag: tissue


title: MBSF7-rna


title: MB4141-rna


title: MB4037-rna


title: MB4036-rna

value: F

value: WNT


title: Whole_blood_LRFI_24_4307_0_9

description: castrated


title: Whole_blood_LRFI_24_3357_-5

description: castrated


title: Whole_blood_HRFI_30_3386_0_13

description: castrated

value: HRFI