title: SW480, shEIF2B1, induced, repl 4 [2B1_3A_plus_Tot_4]

description:
2B1_3A_plus_Tot_4
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MNase_seq_Figure_6_figure_supplement_1_16909_sir2_fun30_G1

status: Public on Jan 09 2025

type: SRA

city: Seattle

first: Antonio

last: Bedalov

state: WA

address: 1100 Fairview Ave N

department: Translational Science and Therapeutics Division

country: USA

institute: Fred Hutch Cancer Center

description: Library name: Figure_6_figure_supplement_1_16909_G1_Mnase_20U_AA20_S9

library_source: genomic

data_processing:
align with bwa using the -n option set to 1; this option specifies that, if a read can be aligned to more than one genomic locations, it is aligned to just one of those location, and that one is chosen at random
picard CleanSam
picard SortSam
picard FixMateInformation
picard AddOrReplaceReadGroups
Assembly: sacCer3
Supplementary files format and content: bedgraph; includes coverage of only library inserts that are 151-200 base pairs; the entire fragment is inferred; e.g if there are two 50 bp reads that define the borders of a 170 bp library insert, the entire 170 bp insert will be reflected in the coverage

molecule: genomic DNA

organism: Saccharomyces cerevisiae

extract_protocol:
MNase seq  We carried out MNase-Seq as previously described (Foss 2019). Briefly, cells grown to log phase in rich medium, Yeast Peptone Agar with 2% glucose (YEPD), from an overnight 25 mL culture were synchronized with 3 μM alpha-factor for 1.5 hrs. at 30 ° C. Arrested cells were crosslinked with 1% formaldehyde for 30 min at room temperature water bath with shaking. Formaldehyde was quenched with 125 mM glycine and cells were centrifuged at 3000 rpm for 5 min. Cells were washed twice with water and resuspended in 1.5 mL Buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) with 1 mM beta-mercaptoethanol (1.1 μL of 14.3 M beta-mercaptoethanol diluted 1:10 in Buffer Z) per 25 mL culture. Cells were treated with 100 μL 20 mg/ mL zymolyase at 30 ° C for 20–30 min. Spheroplasts were centrifuged at 5000 rpm for 10 min and resuspended in 5 mL NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) supplemented with 500 μM spermidine, 1 mM beta-mercaptoethanol and 0.075% NP-40. Nuclei were aliquoted in tubes with varying concentrations of micrococcal nuclease (Worthington), mixed via tube inversion, and incubated at room temperature for 20 mins. Chromatin digested with 1.9 U– 7.5 U micrococcal nuclease per 1/5th of spheroplasts from a 25 mL culture yielded appropriate mono-, di-, tri- nucleosome protected fragments for next-generation sequencing. Digestion was stopped with freshly made 5x stop buffer (5% SDS, 50 mM EDTA) and proteinase K was added (0.2 mg/ml final concentration) for an overnight incubation at 65 ° C to reverse crosslinking. DNA was extracted with phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Sequencing libraries were prepared as described below for ChEC.  Chromatin Endogenous Cleavage (ChEC)  ChEC-seq was carried out as previously described (Foss et al. 2019; Foss et al. 2021). Briefly, MNase was activated by addition of CaCl2 to cells that were permeabilized with digitonin. DNA was extracted from cells using phenol and chloroform, precipitated using salt and ethanol and used to construct sequencing libraries without size fractionation. Cells were centrifuged at 1,500 x g for 2 mins, and washed twice in cold Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA) without additives. Washed cells were carefully resuspended in 570 μL Buffer A with additives (0.2 mM spermidine, 0.5 mM spermine, 1 mM PMSF, ½ cOmplete ULTRA protease inhibitors tablet, Roche, per 5 mL Buffer A) and permeabilized with 0.1% digitonin in 30 ° C water bath for 5 min. Permeabilized cells were cooled at room temperature for 1 min and 1/5th of cells were transferred in a tube with freshly made 2x stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA)/1% SDS solution for undigested control. Micrococcal nuclease was activated with 5.5 μL of 200 mM CaCl2 at various times (5 mins, and 10 mins) and the reaction stopped with 2x stop buffer/1% SDS. Once all time points were collected, proteinase K was added to each of the collected time points and incubated at 55 ° C water bath for 30 mins. DNA was extracted using phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Library was prepared as previously described used using total DNA, without any fragment size selection (Foss et al. 2019; Foss et al. 2021).  Chromatin immunoprecipitation followed by sequencing (ChIP-seq)  Cells were grown to log phase in rich medium, YEPD (1% Yeast Extract, 2% Peptone, 2% glucose), synchronized with 3 μM alpha-factor for 1.5 hrs. at 30°C. G1 arrested cells were released into 200 mM hydroxyurea (HU) YEPD and grown for 1hr. Cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 125 mM glycine for 5 minutes, and centrifuged at 3000 rpm for 5 minutes. Cell pellets were washed three times with TBS (20 mM Tris-HCl, pH 7.6, 150 mM NaCl). Cell pellets were flash frozen.  Chromatin Preparation  The cell pellet was thawed in 300 μL Breaking Buffer (100 mM Tris- HCl, pH 8.0, 20% Glycerol) supplemented with protease inhibitors (Pierce Protease inhibitor EDTA-free tablets [Thermo Scientific A32965] and 1 mM PMSF). 300 μL acid-washed glass beads were added to the mixture. Cells were lysed using a BioSpec Mini-Bead-beater in a cold room: 5x 30-second pulses with 1-minute on ice between rounds. The supernatant was transferred to a new tube and added 600 μL FA buffer (50 mM Hepes-KOH, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate) with protease inhibitors. The chromatin was sonicated with a F60 Sonic Dismembrator 10 times for 10 seconds at setting 4, with 1-minute rests on ice between rounds. The sonicated chromatin was centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was transferred and spun again at 14,000 rpm for 15 minutes at 4°C.  Immunoprecipitation (IP)  For IP, 20 μL of Dynabeads Protein G beads (Invitrogen 10004D) were washed three times with 500 μL PBS-T (0.8% NaCl, 0.144% Na2HPO4, 0.02% KCl, 0.024% KH2PO, 0.1% Tween 20, pH 7.1), resuspended in PBS-T, and incubated with 5 μL of FLAG M2 (Sigma F1804) antibody for 60 minutes at room temperature. The antibody-conjugated beads were washed and resuspended in 20 μL FA buffer with protease inhibitors. IP chromatin samples (1 μg) were brought up to 400 μL with FA buffer with protease inhibitors and incubated with antibody- conjugated beads for 90 minutes at room temperature. Beads were washed three times with FA buffer with protease inhibitors, twice with FA-HS buffer (50 mM Hepes-KOH, pH 7.6, 500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate with protease inhibitors), and once with RIPA buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate, 5 mM EDTA with protease inhibitors). Input sample was generated by adding 0.1 μg chromatin in 40 μL FA buffer to 40 μL 2X Stop buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS, 2% Tween 20) and reversed crosslinks by overnight incubation at 65°C.  Elution and Crosslink Reversal  Bound chromatin was eluted by incubating the beads in 80 μL 2X Stop buffer at 75°C for 10 minutes. The eluate was collected, and crosslinks were reversed by overnight incubation at 65°C. The IP and Input were treated with 4 μL of 20 mg/mL RNase A and incubated at 55°C for 1 hour, followed by 4 μL of 20 mg/mL Proteinase K and incubation at 55°C for at least 3 hours. DNA was purified using a MinElute PCR Purification Kit  Paired-Ends NGS  Chromatin immunoprecipitation samples were prepared for sequencing following the protocol for the Kapa Hyper Kit. For Input samples, 2 ng were processed for End Repair. For IP samples, the entire volume was used. PCR amplification was performed with 10- 14 cycles based on DNA quantity.  Quantification of rDNA replication using the thymidine analogue EdU  Cells were grown to log phase in rich YEPD medium from a 50 mL culture and synchronized in G1 using 3 μM alpha factor for 2 hours at 30°C. Using 500 μg/mL pronase, arrested cells were released into rich media containing 200 mM hydroxyurea and 130 μM EdU (5-Ethynyl-2’- deoxyuridine). Cells were harvested in G1 and after 60 minutes released into HU. DNA was extracted using YeaStar Genomic DNA Kit (Zymo Research) and sonicated. Separate sequencing primers were ligated to each of the samples. Samples were pooled together, and subjected to click chemistry reaction in 10 mM Tris pH 7.4, 50mM NaCl, 20 μM biotin-TEG azide (Vector Laboratories), 5mM THPTA (tris-hydroxypropyltriazolylmethylamine) (Vector Laboratories), 1mM copper sulfate, and 15 mM sodium ascorbate (total volume 200 μL). Click reaction proceeded for 30 minutes at room temperature prior to DNA ethanol precipitation. Streptavidin magnetic beads (New England Biolabs) were washed 3 times with B&W buffer I (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween20, 0.5% SDS) before adding 100 μL of bead suspension to 180 μL DNA plus B&W buffer I. After a 30-minute incubation at room temperature while gently inverting tubes, bead bound DNA was washed with 200 μL B&W buffer I followed by 100 μL Stringency wash buffer (0.1% SDS, 0.1% SSC buffer) and 200 μL Wash Buffer II (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween 20). DNA was eluted off the beads by denaturation at 98°C for 5 minutes, followed by cooling at 25°C. Edu-labeled DNA was PCR amplified post-pulldown and subjected to sequencing.  Hydoxyurea ChEC time course experiment  Cells grown in synthetic media to logarithmic phase were arrested in G1 using alpha factor for 90 minutes, centrifuged, washed in water to remove alpha factor, and released into media containing 200 mM Hydroxyurea. Cells were harvested in G1 and at indicated time points following release into HU-containing media, chilled at 4 degrees and analyzed by ChEC as described above.
MNase seq  We carried out MNase-Seq as previously described (Foss 2019). Briefly, cells grown to log phase in rich medium, Yeast Peptone Agar with 2% glucose (YEPD), from an overnight 25 mL culture were synchronized with 3 μM alpha-factor for 1.5 hrs. at 30 ° C. Arrested cells were crosslinked with 1% formaldehyde for 30 min at room temperature water bath with shaking. Formaldehyde was quenched with 125 mM glycine and cells were centrifuged at 3000 rpm for 5 min. Cells were washed twice with water and resuspended in 1.5 mL Buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) with 1 mM beta-mercaptoethanol (1.1 μL of 14.3 M beta-mercaptoethanol diluted 1:10 in Buffer Z) per 25 mL culture. Cells were treated with 100 μL 20 mg/ mL zymolyase at 30 ° C for 20–30 min. Spheroplasts were centrifuged at 5000 rpm for 10 min and resuspended in 5 mL NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) supplemented with 500 μM spermidine, 1 mM beta-mercaptoethanol and 0.075% NP-40. Nuclei were aliquoted in tubes with varying concentrations of micrococcal nuclease (Worthington), mixed via tube inversion, and incubated at room temperature for 20 mins. Chromatin digested with 1.9 U– 7.5 U micrococcal nuclease per 1/5th of spheroplasts from a 25 mL culture yielded appropriate mono-, di-, tri- nucleosome protected fragments for next-generation sequencing. Digestion was stopped with freshly made 5x stop buffer (5% SDS, 50 mM EDTA) and proteinase K was added (0.2 mg/ml final concentration) for an overnight incubation at 65 ° C to reverse crosslinking. DNA was extracted with phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Sequencing libraries were prepared as described below for ChEC.  Chromatin Endogenous Cleavage (ChEC)  ChEC-seq was carried out as previously described (Foss et al. 2019; Foss et al. 2021). Briefly, MNase was activated by addition of CaCl2 to cells that were permeabilized with digitonin. DNA was extracted from cells using phenol and chloroform, precipitated using salt and ethanol and used to construct sequencing libraries without size fractionation. Cells were centrifuged at 1,500 x g for 2 mins, and washed twice in cold Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA) without additives. Washed cells were carefully resuspended in 570 μL Buffer A with additives (0.2 mM spermidine, 0.5 mM spermine, 1 mM PMSF, ½ cOmplete ULTRA protease inhibitors tablet, Roche, per 5 mL Buffer A) and permeabilized with 0.1% digitonin in 30 ° C water bath for 5 min. Permeabilized cells were cooled at room temperature for 1 min and 1/5th of cells were transferred in a tube with freshly made 2x stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA)/1% SDS solution for undigested control. Micrococcal nuclease was activated with 5.5 μL of 200 mM CaCl2 at various times (5 mins, and 10 mins) and the reaction stopped with 2x stop buffer/1% SDS. Once all time points were collected, proteinase K was added to each of the collected time points and incubated at 55 ° C water bath for 30 mins. DNA was extracted using phenol/chloroform and precipitated with ethanol. Micrococcal nuclease digestion was analyzed via gel electrophoresis prior to proceeding to library preparation. Library was prepared as previously described used using total DNA, without any fragment size selection (Foss et al. 2019; Foss et al. 2021).  Chromatin immunoprecipitation followed by sequencing (ChIP-seq)  Cells were grown to log phase in rich medium, YEPD (1% Yeast Extract, 2% Peptone, 2% glucose), synchronized with 3 μM alpha-factor for 1.5 hrs. at 30°C. G1 arrested cells were released into 200 mM hydroxyurea (HU) YEPD and grown for 1hr. Cells were crosslinked with 1% formaldehyde for 30 min at room temperature, quenched with 125 mM glycine for 5 minutes, and centrifuged at 3000 rpm for 5 minutes. Cell pellets were washed three times with TBS (20 mM Tris-HCl, pH 7.6, 150 mM NaCl). Cell pellets were flash frozen.  Chromatin Preparation  The cell pellet was thawed in 300 μL Breaking Buffer (100 mM Tris- HCl, pH 8.0, 20% Glycerol) supplemented with protease inhibitors (Pierce Protease inhibitor EDTA-free tablets [Thermo Scientific A32965] and 1 mM PMSF). 300 μL acid-washed glass beads were added to the mixture. Cells were lysed using a BioSpec Mini-Bead-beater in a cold room: 5x 30-second pulses with 1-minute on ice between rounds. The supernatant was transferred to a new tube and added 600 μL FA buffer (50 mM Hepes-KOH, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate) with protease inhibitors. The chromatin was sonicated with a F60 Sonic Dismembrator 10 times for 10 seconds at setting 4, with 1-minute rests on ice between rounds. The sonicated chromatin was centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was transferred and spun again at 14,000 rpm for 15 minutes at 4°C.  Immunoprecipitation (IP)  For IP, 20 μL of Dynabeads Protein G beads (Invitrogen 10004D) were washed three times with 500 μL PBS-T (0.8% NaCl, 0.144% Na2HPO4, 0.02% KCl, 0.024% KH2PO, 0.1% Tween 20, pH 7.1), resuspended in PBS-T, and incubated with 5 μL of FLAG M2 (Sigma F1804) antibody for 60 minutes at room temperature. The antibody-conjugated beads were washed and resuspended in 20 μL FA buffer with protease inhibitors. IP chromatin samples (1 μg) were brought up to 400 μL with FA buffer with protease inhibitors and incubated with antibody- conjugated beads for 90 minutes at room temperature. Beads were washed three times with FA buffer with protease inhibitors, twice with FA-HS buffer (50 mM Hepes-KOH, pH 7.6, 500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate with protease inhibitors), and once with RIPA buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.5% NP-40, 0.5% Sodium deoxycholate, 5 mM EDTA with protease inhibitors). Input sample was generated by adding 0.1 μg chromatin in 40 μL FA buffer to 40 μL 2X Stop buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS, 2% Tween 20) and reversed crosslinks by overnight incubation at 65°C.  Elution and Crosslink Reversal  Bound chromatin was eluted by incubating the beads in 80 μL 2X Stop buffer at 75°C for 10 minutes. The eluate was collected, and crosslinks were reversed by overnight incubation at 65°C. The IP and Input were treated with 4 μL of 20 mg/mL RNase A and incubated at 55°C for 1 hour, followed by 4 μL of 20 mg/mL Proteinase K and incubation at 55°C for at least 3 hours. DNA was purified using a MinElute PCR Purification Kit  Paired-Ends NGS  Chromatin immunoprecipitation samples were prepared for sequencing following the protocol for the Kapa Hyper Kit. For Input samples, 2 ng were processed for End Repair. For IP samples, the entire volume was used. PCR amplification was performed with 10- 14 cycles based on DNA quantity.  Quantification of rDNA replication using the thymidine analogue EdU  Cells were grown to log phase in rich YEPD medium from a 50 mL culture and synchronized in G1 using 3 μM alpha factor for 2 hours at 30°C. Using 500 μg/mL pronase, arrested cells were released into rich media containing 200 mM hydroxyurea and 130 μM EdU (5-Ethynyl-2’- deoxyuridine). Cells were harvested in G1 and after 60 minutes released into HU. DNA was extracted using YeaStar Genomic DNA Kit (Zymo Research) and sonicated. Separate sequencing primers were ligated to each of the samples. Samples were pooled together, and subjected to click chemistry reaction in 10 mM Tris pH 7.4, 50mM NaCl, 20 μM biotin-TEG azide (Vector Laboratories), 5mM THPTA (tris-hydroxypropyltriazolylmethylamine) (Vector Laboratories), 1mM copper sulfate, and 15 mM sodium ascorbate (total volume 200 μL). Click reaction proceeded for 30 minutes at room temperature prior to DNA ethanol precipitation. Streptavidin magnetic beads (New England Biolabs) were washed 3 times with B&W buffer I (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween20, 0.5% SDS) before adding 100 μL of bead suspension to 180 μL DNA plus B&W buffer I. After a 30-minute incubation at room temperature while gently inverting tubes, bead bound DNA was washed with 200 μL B&W buffer I followed by 100 μL Stringency wash buffer (0.1% SDS, 0.1% SSC buffer) and 200 μL Wash Buffer II (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05% Tween 20). DNA was eluted off the beads by denaturation at 98°C for 5 minutes, followed by cooling at 25°C. Edu-labeled DNA was PCR amplified post-pulldown and subjected to sequencing.  Hydoxyurea ChEC time course experiment  Cells grown in synthetic media to logarithmic phase were arrested in G1 using alpha factor for 90 minutes, centrifuged, washed in water to remove alpha factor, and released into media containing 200 mM Hydroxyurea. Cells were harvested in G1 and at indicated time points following release into HU-containing media, chilled at 4 degrees and analyzed by ChEC as described above.

tag: cell line

tag: cell type

value: haploid

tag: genotype

value: Mat A, his3, leu2, met15, ura3, fun30::KanMX, sir2::LEU (#490), hml::hyg


title: SW480, shEIF2B1, uninduced, repl 3 [2B1_3A_min_Tot_3_c]

city: Berlin

first: Chiara

last: Giacomelli

address: Robert-Rössle-Str. 10

country: Germany

institute: Max Delbrück Center for Molecular Medicine

description:
2B1_3A_min_Tot_3_c
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv

library_source: transcriptomic

data_processing:
Processing of all ribosome profiling data was carried out using scripts publicly available the https://github.com/Bushell-lab/Ribo-seq GitHub package. All R scripts were carried out using R version 4.3.1.
For cytoplasmic RNA samples: Cutadapt (version 1.18) was used to remove adaptors, trim 3’ bases with quality scores below 20 and discard reads less than 30 bases after trimming. Reads are deduplicated using cd-hit-dup and then UMIs are removed with cutadapt (v 1.18). BBmap (version 38.18) was used to align reads to a FASTA file containing rRNA, tRNA, and mitochondrial encoded RNA reads (GENECODE v38). Transcript isoform and gene level quantification were then carried out using with RSEM (with bowtie2 as alignment algorythm (version 2.3.5.1)). Custom R script and python script were then used to calculate the most abundant transcript per gene, and to create the FASTA to which RPFs were subsequently aligned to.
For Ribosome Protected Fractments (RPFs): Cutadapt (version 1.18) was used to remove adaptors, trim 3’ bases with quality scores below 20 and discard reads less than 30 and more than 50 bases after trimming. Reads are deduplicated using cd-hit-dup and then UMIs are removed with cutadapt (v 1.18). BBmap (version 38.18) was used to align reads to a FASTA file containing rRNA, tRNA, and mitochondrial encoded RNA reads (GENECODE v38). All unaligned reads were then aligned to a filtered protein coding FASTA, containing the most abundant transcript per gene (calculated from cytoplasmic RNA samples). The resulting BAM files were sorted with samtools (version 1.9). The number of reads in which the 5’ end is at each position of every transcript is then counted and the correct offset for each read length is calculated and applied, to collapse all reads into a single frame. Total counts across the entire CDS, excluding the first 20 and last 10 codons are then summed together.
Assembly: GENCODE v38 Protein-coding transcript sequences, filtered to contain only transcripts that have both a 5’ and 3’ UTR, with a CDS equally divisible by 3 and an nUG start codon, a stop codon and that have been manually annotated by HAVANA. All PAR_Y transcripts were also removed.
Supplementary files format and content: Totals_2B1_2B4_NTCcorrected_normalised_counts.csv contains gene-level and transcript-level counts, normalised using the DESeq2 packge Regularized log transformation (rlog(dds)). Only genes with an average of 10 reads across all samples are included
Supplementary files format and content: RPFs_2B1_2B4_NTCcorrected_normalised_counts.csv contains gene-level and transcript-level counts aligned to the coding sequences and summed as described above. Normalisation is done using the regularised log transformation formula (rlog(dds)) from DESeq2 package. Only genes with an average of 10 reads across all samples are included

molecule: cytoplasmic RNA

organism: Homo sapiens

source_name: Colon

growth_protocol: SW480 cells were cultured in RPMI 1640 (#21875091, Thermo Fisher Scientific), supplemented with 10 % heat-inactivated FBS (#FBS-11A, Capricorn Scientific) and 1% penicillin/streptomycin (#P4333, Sigma).

extract_protocol:
Pellets were resuspended in non-denaturing lysis buffer (15 mM Tris-Cl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1% Triton X-100, 0.05% Tween-20, 2% n-Dodecyl-beta-Maltoside Detergent (DDM), 0.5 mM DTT, 5 mM NaF, 1x cOmplete mini (#04693124001, Roche)). For RPFs: Lysates were digested with 8 μl of RNAseI (#AM2295, Lot# 00791236, Ambion) at 22°C for 20 min. Digestion was stopped with 16 μl of SUPERaseIn (#AM2696, Invitrogen). Digested lysates were run on a 10-50% sucrose gradients where fractions corresponding to monosomes and disomes were collected. RNA from these fractions was extracted by acid phenol-chloroform extractions, further processed to select for ribosome protected frament (RPF) size, treated with T4 Polynucleotide Kinase (NEB M0201S) and size checked on an Agilent Bioanalyzer small RNA chip. For total cytoplasmic RNA: prior to digesting the lysate for RPF extraction, an aliquot was kept aside and mixed with 1 ml of TRIzol™ Reagent (Invitrogen, 15596026). RNA extraction from TRIzol was performed according to manufacturer's instruction, with a further Ethanol precipitation to eliminate all impurities. Average eRIN value of total cytoplasmic RNAs was 9.7, as checked by RNA screen tape on a TapeStation (Agilent)
Total cytoplasmic RNAs were depleted of rRNA with RiboCop v2 (Lexogen) and then libraries were prepared using the CORALL kit (Lexogen), all following the manufacturer’s instructions. 900 μg of total cytoplasmic RNAs were used as input for the RiboCop. 13 PCR cycles were used in the library amplification stage. For RPFs: Libraries were prepared with the NEXTflex small RNA kit v3 (Perkin Elmer) following the manufacturer’s instructions, using 10.5 ng of input and 11 PCR cycles in the amplification step. Alternative step F and Step H2 were used.

treatment_protocol: SW480 cells  transduced with inducible non-targeting control (CTRL), EIF2B1, or EIF2B4 shRNAs were grown in the absence (DMSO) or presence of 1 µg/ml doxycycline for five days. Two 15 cm dishes per condition were then harvested in cold PBS, washed twice in cold PBS, and the resulting pellet was snap-frozen in liquid nitrogen. One additional 15 cm dish per condition was harvested in parallel to check efficient knockdown by immunoblotting. The experiment was performed four times in separate weeks.

tag: tissue

value: Colon

tag: shRNA

value: inducible shEIF2B1

value: Adenocarcinoma; Colorectal; Dukes' type B

tag: treatment

value: uninduced (DMSO)


title: SW480, shEIF2B1, induced, repl 1 [2B1_3A_plus_Tot_1]

description:
2B1_3A_plus_Tot_1
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv

value: induced (Doxycicline)


title: MNase_seq_Figure_6_figure_supplement_1_16668_sir2_G1

description: Library name: Figure_6_figure_supplement_1_16668_sir2_G1_8U_Mnase_AA37_LP20240426_2_S8

value: Mat a  his3 leu2 met15 ura3 sir2::Kanmx, hml:HYG


title: SW480, shEIF2B1, uninduced, repl 4 [2B1_3A_min_Tot_4]

description:
2B1_3A_min_Tot_4
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: SW480, shEIF2B1, uninduced, repl 2 [2B1_3A_min_Tot_2]

description:
2B1_3A_min_Tot_2
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: SW480, shEIF2B1, uninduced, repl 1 [2B1_3A_min_Tot_1]

description:
2B1_3A_min_Tot_1
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MNase_seq_Figure_6A_17248_fun30_G1_MNASE_16U

description: Library name: Figure_6A_17248_fun30_G1_MNASE_16U_AA21_S2

value: Mat A his3 leu2 met15 ura3m, hml:HYG, fun30::URA


title: MNase_seq_Figure_6A_16909_sir2_fun30_G1_MNASE_16U

description: Library name: Figure_6A_16909_sir2_fun30_G1_MNASE_16U_AA20_S1


title: SW480, shCTRL, induced, repl 4 [NTC_plus_Tot_4]

description:
NTC_plus_Tot_4
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv

value: inducible shCTRL


title: SW480, shCTRL, induced, repl 3 [NTC_plus_Tot_3_c]

description:
NTC_plus_Tot_3_c
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MNase_seq_Figure_6A_16668sir2_lane2

description: Library name: Figure_6A_16668sir2_AF_7_5U_CGATGT_L002


title: SW480, shCTRL, induced, repl 2 [NTC_plus_Tot_2]

description:
NTC_plus_Tot_2
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MNase_seq_Figure_6A_16668sir2_lane1

description: Library name: Figure_6A_16668sir2_AF_7_5U_CGATGT_L001


title: MNase_seq_Figure_6_figure_supplement_1_16535_WT_G1

description: Library name: Figure_6_figure_supplement_1_16535_WT_G1_8U_Mnase_AA36_LP20240426_1_S7

value: Mat A his3 leu2 met15 ura3, hml::HYG


title: SW480, shEIF2B4, uninduced, repl 3 [2B4_2_min_Tot_3_c]

description:
2B4_2_min_Tot_3_c
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv

value: inducible shEIF2B4


title: SW480, shCTRL, uninduced, repl 1 [NTC_min_Tot_1]

description:
NTC_min_Tot_1
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MCM2ChEC_Figure5_figure_supplement_4_17257_HU45m_sir2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17257_HU45m_sir2_fun30_MCM2_Mnase_AA22_S9

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 3xFlag Tag with KanMx,  sir2::LEU, fun30:NAT


title: MCM2ChEC_Figure5_figure_supplement_4_17257_HU60m_sir2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17257_HU60m_sir2_fun30_MCM2_Mnase_AA23_S10


title: SW480, shCTRL, induced, repl 1 [NTC_plus_Tot_1]

description:
NTC_plus_Tot_1
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MCM2ChEC_Figure5_figure_supplement_4_17257_G1_2_sir2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17257_G1_2_sir2_fun30_MCM2_Mnase_AA18_S6


title: MNase_seq_Figure_6A_16535wt_lane1

description: Library name: Figure_6A_16535wt_AF_7_5U_ATCACG_L001


title: SW480, shCTRL, uninduced, repl 2 [NTC_min_Tot_2]

description:
NTC_min_Tot_2
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: LUNG_FIBRO_WT3

city: New Haven

first: Andrés

last: Hidalgo

address: 10 Amistad Street

institute: Yale University

data_processing:
FastQ files for each sample were obtained using CASAVA v1.8 software (Illumina)
Filtering of low quality reads and removal of adapters with cutadapt v1.7.1
Alignment and quantification with RSEM v1.7.1
Normalization with Limma’s voom v3.46
Assembly: mm10
Supplementary files format and content: normalized TPMs from each tissue

molecule: total RNA

organism: Mus musculus

source_name: Lung

extract_protocol:
Lung and Skin macrophages and fibroblasts were FACS sorted using a cocktail of fluorescent antibodies (Lung interstitial macrophages: DAPI-CD45+CD68+F4/80+CD11c-CD11bhi; Skin macrophages: DAPI-CD45+CD11b+CD68+F4/80+; Lung fibroblasts: DAPI-CD45-CD31-EPCAM-PDGFRA+;  Skin fibroblasts: DAPI-CD45-CD31-EPCAM-CD90.1+PDGFRA+) with typical purities > 95%. RNA was extracted ussing the RNAeasy plus micro kit from QUIAGEN
cDNA amplification from neutrophil RNA and generation of index-tagged sequencing libraries were carried out using the Ovation Single Cell RNA-Seq System (NuGEN Technologies, San Carlos CA). Libraries were quantified using a Quant-iTTM dsDNA HS assay with the Q-bit fluorometer (Life Technologies, Carlsbad, California). Average library size and size distribution were determined using a High Sensitivity DNA assay in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA). Libraries were normalized to 10nM using Tris-Cl 10mM, pH8.5 with 0.1% Tween 20. Libraries were applied to an Illumina flow cell for cluster generation (True Seq SR Cluster Kit V2 cBot) and 61 nt long, single-end reads were generated on a Genome Analyzer IIx, using the TruSeq SBS Kit v5 (Illumina) and following the standard RNA sequencing protocol

treatment_protocol: all mice were Injected intraperitoneally with 10 ug/kg of diphtheria toxin for 7 days

value: Lung

tag: strain

value: Fibroblasts

value: Mrp8_WT; Rosa26_DTR


title: MCM2ChEC_Figure5_figure_supplement_4_17257_HU15m_sir2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17257_HU15m_sir2_fun30_MCM2_Mnase_AA19_S7


title: SW480, shCTRL, uninduced, repl 3 [NTC_min_Tot_3_c]

description:
NTC_min_Tot_3_c
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv


title: MCM2ChEC_Figure5_figure_supplement_4_17256_HU60m_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_HU60m_fun30_MCM2_Mnase_AA14_S14

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 MNase 3xFlag Tag with KanMx, fun30::NAT


title: LUNG_FIBRO_NP3

value: Mrp8_CRE; Rosa26_DTR

value: Injected intraperitoneally with 10 ug/kg of diphtheria toxin for 7 days


title: MCM2ChEC_Figure5_figure_supplement_4_17256_HU90m_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_HU90m_fun30_MCM2_Mnase_AA15_S16

value: haploid


title: MCM2ChEC_Figure5_figure_supplement_4_17257_HU30m_sir2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17257_HU30m_sir2_fun30_MCM2_Mnase_AA20_S8


title: LUNG_FIBRO_NP1


title: MCM2ChEC_Figure5_figure_supplement_4_17256_HU45m_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_HU45m_fun30_MCM2_Mnase_AA13_S13


title: MNase_seq_Figure_6A_16535wt_lane2

description: Library name: Figure_6A_16535wt_AF_7_5U_ATCACG_L002


title: SW480, shCTRL, uninduced, repl 4 [NTC_min_Tot_4]

description:
NTC_min_Tot_4
Totals_2B1_2B4_NTCcorrected_normalised_counts.csv

value: Colon


title: LUNG_FIBRO_WT2


title: LUNG_MAC_NP2

value: Macrophages


title: LUNG_MAC_NP3


title: MCM2ChEC_Figure5_figure_supplement_4_17256_HU30m_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_HU30m_fun30_MCM2_Mnase_AA12_S12


title: MCM2ChEC_Figure5_figure_supplement_4_17256_G1-2_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_G1-2_fun30_MCM2_Mnase_AA10_S9


title: MCM2ChEC_Figure5_figure_supplement_4_16769_HU90m_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_HU90m_sir2_MCM2_Mnase_AA09_S15

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 3xFlag Tag with KanMx,  sir2,:LEU


title: LUNG_MAC_NP1


title: LUNG_FIBRO_WT1


title: MCM2ChEC_Figure5_figure_supplement_4_16769_HU60m_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_HU60m_sir2_MCM2_Mnase_AA07_S8


title: LUNG_FIBRO_NP2


title: LUNG_MAC_WT3


title: SKIN_MFIBRO_WT5

source_name: Skin

value: Skin


title: MCM2ChEC_Figure5_figure_supplement_4_16747_HU60m_WT_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16747_HU60m_WT_MCM2_Mnase_AA22_S3

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 MNase 3xFlag Tag with KanMx


title: MCM2ChEC_Figure5_figure_supplement_4_16769_HU30m_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_HU30m_sir2_MCM2_Mnase_AA05_S6


title: MCM2ChEC_Figure5_figure_supplement_4_16769_G1-2_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_G1-2_sir2_MCM2_Mnase_AA03_S4


title: LUNG_MAC_WT1


title: SKIN_MAC_WT1


title: MCM2ChEC_Figure5_figure_supplement_4_16747_HU30m_WT_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16747_HU30m_WT_MCM2_Mnase_AA18_S1


title: SKIN_MAC_NP3


title: SKIN_MAC_WT3


title: MCM2ChEC_Figure5_figure_supplement_4_16747_HU15m_WT_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16747_HU15m_WT_MCM2_Mnase_AA16_S16


title: LUNG_MAC_WT2


title: MCM2ChEC_Figure5_figure_supplement_4_16769_HU45m_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_HU45m_sir2_MCM2_Mnase_AA06_S7


title: MCM2ChEC_Figure5_figure_supplement_4_16769_HU15m_sir2_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16769_HU15m_sir2_MCM2_Mnase_AA04_S5


title: MCM2ChEC_Figure5_figure_supplement_4_17256_HU15m_fun30_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_17256_HU15m_fun30_MCM2_Mnase_AA11_S11


title: SKIN_FIBRO_WT5


title: SKIN_MAC_NP5


title: SKIN_MAC_NP2


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_HU_IP_ChIP_sonic_dup_2

description: Library name: Figure5_figure_supplement_2_17558_WT_HU_IP_ChIP_sonic_AA19_KLP20240722_dup_2_S14

value: Mat A his3 leu2 met15 ura3, hml, MCM2 6xGLY 3xFlag KanMX


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_HU_IP_ChIP_sonic

description: Library name: Figure5_figure_supplement_2_17558_WT_HU_IP_ChIP_sonic_AA02_KLP20240717_2_S2


title: MCM2ChEC_Figure5_figure_supplement_4_16747_HU45m_WT_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16747_HU45m_WT_MCM2_Mnase_AA20_S2


title: SKIN_FIBRO_WT4


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_HU_Input_ChIP_sonic_dup_6

description: Library name: Figure5_figure_supplement_2_17558_WT_HU_Input_ChIP_sonic_AA23_KLP20240722_dup_6_S18


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_HU_Input_ChIP_sonic

description: Library name: Figure5_figure_supplement_2_17558_WT_HU_Input_ChIP_sonic_AA06_KLP20240717_6_S6


title: MCM2ChEC_Figure5_figure_supplement_4_16747_G1_1_WT_MCM2_Mnase

description: Library name: Figure5_figure_supplement_4_16747_G1_1_WT_MCM2_Mnase_AA15_S15


title: SKIN_MAC_NP1


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_G1_Input_ChIP_sonic_dup_5

description: Library name: Figure5_figure_supplement_2_17558_WT_G1_Input_ChIP_sonic_AA22_KLP20240722_dup_5_S17


title: SKIN_FIBRO_WT3


title: SKIN_FIBRO_NP4


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_G1_Input_ChIP_sonic

description: Library name: Figure5_figure_supplement_2_17558_WT_G1_Input_ChIP_sonic_AA05_KLP20240717_5_S5


title: SKDT9

extract_protocol:
Whole RNA was obtained subsequently using mechanical disruption (Polytron PT 6100; Kinematica) and chloroform extraction and cleaned up using silica-based spin columns (Qiagen) according to manufacturer instructions. RNA quality was checked using capillary electrophoresis (Agilent).
Samples were submitted for whole RNA next generation sequencing in the Genomics Unit of CNIC. 200 ng of total RNA was used to generate barcoded RNA sequencing libraries using the NEBNext Ultra RNA Library preparation kit (New England Biolabs). Libraries were sequenced with HiSeq2500 (Illumina) to generate 50-nucleotide single reads, with minimum of eight million reads per sample. FastQ files for each sample were obtained using CASAVA v1.8 software (Illumina). Reads were further processed using the CASAVA package (Illumina) to demultiplex reads according to adapter indexes and to produce FastQ files. Read quality was determined with the application FastQC. For data analysis, sequencing adaptor contaminations were removed from reads using Cutadapt v1.7.1, and the resulting reads were mapped on the transcriptome (GRCm38 Ensembl gene-build 84) and quantified using RSEM v1.2.20 and Kallisto v0.43.1.

treatment_protocol: Mice were treated with 3 µg/g mouse bodyweight of either anti-Ly6G (neutrophil depleted) or control IgG (nondepleted) antibodies for two consecutive days, in normal light-controlled mouse facility or 12 h inverted light cabinets. In the morning of the third day, mice were bled from the cheek just before the lung harvest to obtain blood samples for confirmation of neutrophil depletion using a standard hemocytometer (ABX Pentra 80; Horiba), as well as flow cytometry. Blood, bone marrow, lung, samples were collected every four hours (ZT1-5-9-13-17-21) from 3 mice per group. In the Mrp8CRE; iDTR model, mice were injected intraperitoneally with 10 μg/kg of diphtheria toxin (DT; Calbiochem) for two consecutive days, and tissues were collected on the third day. Ear skins were harvested after perfusion of mice with saline at ZT1-3-4-5-7-9-10-12-13-15-16-18-19-21-22-24 from one mouse per group and snap-frozen in 1 ml of TRIzol each

tag: mouse

value: DT

tag: time

value: ZT 16

tag: neutropenia model

value: DT depletion

value: Mrp8CRE; iDTR


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_G1_IP_ChIP_sonic_dup_1

description: Library name: Figure5_figure_supplement_2_17558_WT_G1_IP_ChIP_sonic_AA18_KLP20240722_dup_1_S13


title: MCM2ChEC_Figure5_figure_supplement_2_17558_WT_G1_IP_ChIP_sonic

description: Library name: Figure5_figure_supplement_2_17558_WT_G1_IP_ChIP_sonic_AA01_KLP20240717_1_S1


title: SKIN_FIBRO_NP2


title: EdU_input_Figure4F_17281_fun30

description: Library name: Figure4F_17281_fun30_1hr_EdU_input_AA25_S6


title: SKDT8

value: ZT 15


title: SKDT4

value: ZT 9


title: SKIN_FIBRO_NP5

value: Fibroblasts


title: EdU_input_Figure4F_17279_sir2_fun30

description: Library name: Figure4F_17279_sir2_fun30_1hr_EdU_input_AA22_S5


title: EdU_Figure4F_17279_sir2_fun30

description: Library name: Figure4F_17279_sir2_fun30_1hr_EdU_AA22_S5


title: SKDT6

value: ZT 12


title: SKDT5

value: ZT 10


title: SKDT7

value: ZT 13


title: EdU_input_Figure4F_17271_sir2

description: Library name: Figure4F_17271_sir2_1hr_EdU_input_AA18_S3

value: haploid


title: SKDT3

value: ZT 7


title: SKDT16

value: ZT 3


title: EdU_Figure4F_17271_sir2

description: Library name: Figure4F_17271_sir2_1hr_EdU_AA18_S3


title: EdU_input_Figure4F_17265_WT

description: Library name: Figure4F_17265_WT_1hr_EdU_input_AA13_S1


title: EdU_Figure4F_17265_WT

description: Library name: Figure4F_17265_WT_1hr_EdU_AA13_S1


title: SKDT2

value: ZT 5


title: EdU_Figure4F_17281_fun30

description: Library name: Figure4F_17281_fun30_1hr_EdU_AA25_S6


title: SKDT15

value: Skin

value: ZT 1


title: MCM2ChEC_Figure1_figure_supplement1_sir2

description: Library name: Figure1_figure_supplement1_sir2_07_16769_MCM2sir2_5mAF_AA19_GTGAAA_L001

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 3xFlag Tag with KanMx,  sir2,:LEU


title: SKDT14

value: DT

value: ZT 24


title: MCM2ChEC_Fig5C_17257_HU_90m_sir2_fun30

description: Library name: Fig5C_17257_HU_90m_sir2_fun30_ChEC_5m_AA22_S19

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 3xFlag Tag with KanMx,  sir2::LEU, fun30:NAT


title: MCM2ChEC_Fig5C_17257_HU_60m_sir2_fun30

description: Library name: Fig5C_17257_HU_60m_sir2_fun30_ChEC_5m_AA16_S11


title: MCM2ChEC_Figure1_figure_supplement1_WT

description: Library name: Figure1_figure_supplement1_WT_03_16747_MCM2_5mAF_AA25_ACTGAT_L001

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 MNase 3xFlag Tag with KanMx


title: SKDT13

value: ZT 22

value: DT depletion

value: Mrp8CRE; iDTR


title: SKDT12

value: ZT 21


title: SKDT11

value: ZT 19


title: SKDT10

value: ZT 18


title: MCM2ChEC_Fig5C_17257_HU_30m_sir2_fun30

description: Library name: Fig5C_17257_HU_30m_sir2_fun30_ChEC_5m_AA10_S9

value: haploid


title: SKCODT9

value: CONTROL

value: ZT 16

value: WT


title: SKCODT5

value: Skin

value: ZT 10


title: SKCODT8

value: ZT 15


title: SKCODT6

value: ZT 12

value: DT


title: MCM2ChEC_Fig5C_17256_HU_30m_fun30

description: Library name: Fig5C_17256_HU_30m_fun30_ChEC_5m_AA09_S21

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 MNase 3xFlag Tag with KanMx, fun30::NAT


title: MCM2ChEC_Fig5C_17256_HU_60m_fun30

description: Library name: Fig5C_17256_HU_60m_fun30_ChEC_5m_AA15_S15


title: MCM2ChEC_Fig5C_17256_HU_45m_fun30

description: Library name: Fig5C_17256_HU_45m_fun30_ChEC_5m_AA12_S14


title: MCM2ChEC_Fig5C_17257_G1-1_sir2_fun30

description: Library name: Fig5C_17257_G1-1_sir2_fun30_ChEC_5m_AA04_S7

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 3xFlag Tag with KanMx,  sir2::LEU, fun30:NAT


title: SKCODT3

value: ZT 7


title: SKCODT2

value: ZT 5

value: DT depletion


title: MCM2ChEC_Fig5C_17256_G1-2_fun30

description: Library name: Fig5C_17256_G1-2_fun30_ChEC_5m_AA03_S12


title: MCM2ChEC_Fig5C_17256_HU_90m_fun30

description: Library name: Fig5C_17256_HU_90m_fun30_ChEC_5m_AA21_S22


title: MCM2ChEC_Fig5C_16769_HU_90m_sir2

description: Library name: Fig5C_16769_HU_90m_sir2_ChEC_5m_AA20_S18

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 3xFlag Tag with KanMx,  sir2,:LEU


title: SKCODT4

value: ZT 9


title: MCM2ChEC_Fig5C_16769_HU_60m_sir2

description: Library name: Fig5C_16769_HU_60m_sir2_ChEC_5m_AA14_S5


title: SKCODT16

value: ZT 3


title: SKCODT15

value: ZT 1


title: MCM2ChEC_Fig5C_16769_HU_45m_sir2

description: Library name: Fig5C_16769_HU_45m_sir2_ChEC_5m_AA11_S4

value: haploid


title: SKCODT14

value: CONTROL

value: ZT 24


title: MCM2ChEC_Fig5C_16769_HU_30m_sir2

description: Library name: Fig5C_16769_HU_30m_sir2_ChEC_5m_AA08_S3


title: MCM2ChEC_Fig5C_16769_HU_15m_sir2

description: Library name: Fig5C_16769_HU_15m_sir2_ChEC_5m_AA05_S2


title: SKCODT12

value: Skin

value: ZT 21

value: WT


title: MCM2ChEC_Fig5C_17257_HU_45m_sir2_fun30

description: Library name: Fig5C_17257_HU_45m_sir2_fun30_ChEC_5m_AA13_S10


title: SKCODT13

value: ZT 22


title: SKDT1

value: DT

value: ZT 4

value: Mrp8CRE; iDTR


title: MCM2ChEC_Fig5C_17257_HU_15m_sir2_fun30

description: Library name: Fig5C_17257_HU_15m_sir2_fun30_ChEC_5m_AA07_S8

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 3xFlag Tag with KanMx,  sir2::LEU, fun30:NAT


title: SKCODT7

value: ZT 13


title: MCM2ChEC_Fig5C_16769_G1-1_sir2

description: Library name: Fig5C_16769_G1-1_sir2_ChEC_5m_AA02_S1


title: SKCODT11

value: ZT 19

value: DT depletion


title: MCM2ChEC_Fig5C_17256_HU_15m_fun30

description: Library name: Fig5C_17256_HU_15m_fun30_ChEC_5m_AA06_S13

value: Mat A his3 leu2 met15 ura3m, hml::HYG, with MCM2 MNase 3xFlag Tag with KanMx, fun30::NAT


title: MCM2ChEC_Fig5B_16747_WT_HU60m

description: Library name: Fig5B_16747_WT_HU60m_ChEC_10m_AA14_S9

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 MNase 3xFlag Tag with KanMx


title: SKCODT10

value: ZT 18


title: SKBMAL8

description:
skin_dt_normalized_counts.csv
SKBMAL8

value: BMAL

value: ZT 15

value: Mrp8CRE; Bmal1-flox


title: MCM2ChEC_Fig5B_16747_WT_HU45m

description: Library name: Fig5B_16747_WT_HU45m_ChEC_10m_AA12_S7

value: haploid


title: SKBMAL6

description:
skin_dt_normalized_counts.csv
SKBMAL6

value: Skin

value: ZT 12


title: SKBMAL5

description:
skin_dt_normalized_counts.csv
SKBMAL5

value: ZT 10


title: MCM2ChEC_Fig5B_16747_WT_HU30m

description: Library name: Fig5B_16747_WT_HU30m_ChEC_10m_AA10_S5


title: MCM2ChEC_Fig5B_16747_WT_HU15m

description: Library name: Fig5B_16747_WT_HU15m_ChEC_10m_AA08_S4


title: SKBMAL7

description:
skin_dt_normalized_counts.csv
SKBMAL7


title: MCM2ChEC_Fig5B_16747_WT_G1

description: Library name: Fig5B_16747_WT_G1_ChEC_10m_AA04_S2


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep5_S

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep5_S_240628

value: Mat A, Fob1::KanMX, YDR110W, 11,C11, hml::HYG, sir2::LEU Final count of rDNA:179 copies)


title: SKBMAL3

description:
skin_dt_normalized_counts.csv
SKBMAL3

value: ZT 7


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep5_G1

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep5_G1_240628


title: SKBMAL16

description:
skin_dt_normalized_counts.csv
SKBMAL16

value: ZT 3


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep4_S

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep4_S_240628


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep3_S

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep3_S_240628


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep4_G1

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep4_G1_240628


title: SKBMAL2

description:
skin_dt_normalized_counts.csv
SKBMAL2

value: ZT 5


title: sort_seq__Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep3_G1

description: Library name: Fig2_fig_suppl_3_179copy_17562_fob1_sir2_rep3_G1_240628


title: SKBMAL15

description:
skin_dt_normalized_counts.csv
SKBMAL15

value: ZT 1


title: sort_seq__Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep2_S

description: Library name: Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep2_S_240628

value: Mat A, Fob1::KanMX, YDR110W, 11,C11, hml::HYG, sir2::LEU Final count of rDNA:177 copies)


title: SKBMAL14

description:
skin_dt_normalized_counts.csv
SKBMAL14

value: ZT 24


title: sort_seq__Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep2_G1

description: Library name: Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep2_G1_240628


title: SKCODT1

value: CONTROL

value: ZT 4

value: DT depletion

value: DT

value: WT


title: SKBMAL13

description:
skin_dt_normalized_counts.csv
SKBMAL13

value: BMAL

value: ZT 22

value: Mrp8CRE; Bmal1-flox


title: SKBMAL11

description:
skin_dt_normalized_counts.csv
SKBMAL11

value: Skin

value: ZT 19


title: sort_seq__Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep1_S

description: Library name: Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep1_S_240628

value: haploid


title: MCM2ChEC_Fig5B_16747_WT_HU90m

description: Library name: Fig5B_16747_WT_HU90m_ChEC_10m_AA16_S11

value: Mat A his3 leu2 met15 ura3m, hml,:HYG, with MCM2 MNase 3xFlag Tag with KanMx


title: sort_seq__Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep1_G1

description: Library name: Fig2_fig_suppl_3_177copy_17561_fob1_sir2_rep1_G1_240628


title: SKBMAL10

description:
skin_dt_normalized_counts.csv
SKBMAL10

value: ZT 18


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep4_G1

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep4_G1_240628

value: Mat A, Fob1::KanMX, YDR110W, 11,C11 fun30::HYG, hml::NAT, sir2::LEU (Final count of rDNA:158 copies)


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep3_S

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep3_S_240628


title: LU9

description:
lung_ab_normalized_counts.csv
LU9

value: Lung

value: depleted

value: ZT 21

value: Antibody depletion

value: anti Ly6G (1A8)


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep3_G1

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep3_G1_240628


title: LU8

description:
lung_ab_normalized_counts.csv
LU8


title: SKBMAL4

description:
skin_dt_normalized_counts.csv
SKBMAL4

value: ZT 9


title: SKBMAL1

description:
skin_dt_normalized_counts.csv
SKBMAL1

value: ZT 4


title: LU6

description:
lung_ab_normalized_counts.csv
LU6

value: WT

value: IgG


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep2_G1

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep2_G1_240628


title: LU5

description:
lung_ab_normalized_counts.csv
LU5


title: sort_seq__Fig2B_swr1_sir2_S_16728_rep1

description: Library name: Fig2B_swr1_sir2_S_16728_rep1_161007

value: Mat A, his3, leu2, met15, ura3, swr1::KanMX, sir2,


title: LU4

description:
lung_ab_normalized_counts.csv
LU4


title: sort_seq__Fig2B_swr1_sir2_G1_16728_rep1

description: Library name: Fig2B_swr1_sir2_G1_16728_rep1_161007


title: LU36

description:
lung_ab_normalized_counts.csv
LU36

value: ZT 17


title: sort_seq__Fig2B_swp_82_sir2_S_17061_rep1

description: Library name: Fig2B_swp_82_sir2_S_17061_rep1_190628

value: Mat A his3 leu2 met15 ura3 swp82::NAT sir2::LEU  colony #2


title: SKBMAL12

description:
skin_dt_normalized_counts.csv
SKBMAL12

value: Skin

value: BMAL

value: Mrp8CRE; Bmal1-flox


title: sort_seq__Fig2B_swp_82_sir2_G1_17061_rep1

description: Library name: Fig2B_swp_82_sir2_G1_17061_rep1_190729

value: haploid


title: LU34

description:
lung_ab_normalized_counts.csv
LU34


title: LU35

description:
lung_ab_normalized_counts.csv
LU35


title: sort_seq__Fig2B_nhp10_sir2_S_16729_rep1

description: Library name: Fig2B_nhp10_sir2_S_16729_rep1_161007

value: Mat A, his3, leu2, met15, ura3, nph10::KanMX, sir2, (#490)


title: LU33

description:
lung_ab_normalized_counts.csv
LU33

value: Lung


title: sort_seq__Fig2B_nhp10_sir2_G1_16729_rep1

description: Library name: Fig2B_nhp10_sir2_G1_16729_rep1_161007


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep4_S

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep4_S_240628

value: Mat A, Fob1::KanMX, YDR110W, 11,C11 fun30::HYG, hml::NAT, sir2::LEU (Final count of rDNA:158 copies)


title: LU32

description:
lung_ab_normalized_counts.csv
LU32


title: LU31

description:
lung_ab_normalized_counts.csv
LU31

value: depleted

value: ZT 13


title: sort_seq__Fig2B_isw2_sir2_G1_16495_rep1

description: Library name: Fig2B_isw2_sir2_G1_16495_rep1_190729

value: mat A, leu2, his3, ura3, isw2,::NAT, his3, leu2, met15, ura3, sir2,::leu (plasmid #490, HindIII)  Transformant 1


title: sort_seq__Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep2_S

description: Library name: Fig2_fig_suppl_3_158copy_17548_fob1_fun30_sir2_rep2_S_240628


title: LU30

description:
lung_ab_normalized_counts.csv
LU30

value: Antibody depletion

value: anti Ly6G (1A8)


title: LU7

description:
lung_ab_normalized_counts.csv
LU7

value: ZT 21

value: WT


title: sort_seq__Fig2B_isw1_sir2_S_16724_rep1

description: Library name: Fig2B_isw1_sir2_S_16724_rep1_161007

value: Mat A, his3, leu2, met15, ura3, isw1::KanMX, sir2, (#490)


title: LU3

description:
lung_ab_normalized_counts.csv
LU3

value: ZT 17


title: sort_seq__Fig2B_isw1_sir2_G1_16724_rep1

description: Library name: Fig2B_isw1_sir2_G1_16724_rep1_161007


title: LU29

description:
lung_ab_normalized_counts.csv
LU29

value: IgG


title: LU28

description:
lung_ab_normalized_counts.csv
LU28


title: sort_seq__Fig2B_irc5_sir2_G1_16723_rep1

description: Library name: Fig2B_irc5_sir2_G1_16723_rep1_161007

value: Mat A, his3, leu2, met15, ura3, irc5::KanMX, sir2, (#490)


title: LU27

description:
lung_ab_normalized_counts.csv
LU27


title: sort_seq__Fig2B_htl1_sir2_S_17059_rep1

description: Library name: Fig2B_htl1_sir2_S_17059_rep1_190628

value: Mat A his3 leu2 met15 ura3 htl1::NAT sir2::LEU  colony #1


title: LU26

description:
lung_ab_normalized_counts.csv
LU26

value: ZT 9


title: sort_seq__Fig2B_fun30_sir2_S_16727_rep4

description: Library name: Fig2B_fun30_sir2_S_16727_rep4_161007

value: haploid

value: Mat A, his3, leu2, met15, ura3, fun30::KanMX, sir2, (#490)


title: sort_seq__Fig2B_htl1_sir2_G1_17059_rep1

description: Library name: Fig2B_htl1_sir2_G1_17059_rep1_190729


title: LU25

description:
lung_ab_normalized_counts.csv
LU25

value: Lung


title: sort_seq__Fig2B_fun30_sir2_S_16727_rep3

description: Library name: Fig2B_fun30_sir2_S_16727_rep3_190628


title: LU22

description:
lung_ab_normalized_counts.csv
LU22


title: sort_seq__Fig2B_fun30_sir2_G1_16727_rep4

description: Library name: Fig2B_fun30_sir2_G1_16727_rep4_161007


title: LU23

description:
lung_ab_normalized_counts.csv
LU23


title: LU24

description:
lung_ab_normalized_counts.csv
LU24


title: LU21

description:
lung_ab_normalized_counts.csv
LU21


title: sort_seq__Fig2B_isw2_sir2_S_16495_rep1

description: Library name: Fig2B_isw2_sir2_S_16495_rep1_190628


title: sort_seq__Fig2B_fun30_sir2_G1_16727_rep3

description: Library name: Fig2B_fun30_sir2_G1_16727_rep3_190729


title: LU20

description:
lung_ab_normalized_counts.csv
LU20

value: ZT 5


title: sort_seq__Fig2B_fun30_S_17248_rep1

description: Library name: Fig2B_fun30_S_17248_rep1_220303

value: Mat A his3 leu2 met15 ura3m, hml:HYG, fun30::URA


title: LU2

description:
lung_ab_normalized_counts.csv
LU2

value: depleted

value: ZT 13


title: sort_seq__Fig2B_fun30_G1_17248_rep1

description: Library name: Fig2B_fun30_G1_17248_rep1_220303


title: LU19

description:
lung_ab_normalized_counts.csv
LU19

value: Antibody depletion


title: sort_seq__Fig2B_fun30_S_16711_rep2

description: Library name: Fig2B_fun30_S_16711_rep2_190628

value: Mat A, his3, leu2, met15, ura3, fun30::KanMX


title: sort_seq__Fig2B_fun30_G1_16711_rep2

description: Library name: Fig2B_fun30_G1_16711_rep2_190729


title: sort_seq__Fig2B_irc5_sir2_S_16723_rep1

description: Library name: Fig2B_irc5_sir2_S_16723_rep1_161007


title: sort_seq__Fig2B_chd1_sir2_S_16725_rep1

description: Library name: Fig2B_chd1_sir2_S_16725_rep1_161007

value: Mat A, his3, leu2, met15, ura3, chd1::KanMX, sir2, (#490)


title: LU17

description:
lung_ab_normalized_counts.csv
LU17

value: WT


title: sort_seq__Fig2B_chd1_sir2_G1_16725_rep1

description: Library name: Fig2B_chd1_sir2_G1_16725_rep1_161007


title: LU18

description:
lung_ab_normalized_counts.csv
LU18


title: LU15

description:
lung_ab_normalized_counts.csv
LU15

value: ZT 1

value: anti Ly6G (1A8)


title: sort_seq__Fig2B_WT_S_14141_rep3

description: Library name: Fig2B_WT_S_14141_rep3_191014

value: Mat A his3 leu2 met15 ura3


title: sort_seq__Fig2B_WT_S_14141_rep2

description: Library name: Fig2B_WT_S_14141_rep2_190729


title: sort_seq__Fig2B_WT_S_14141_rep1

description: Library name: Fig2B_WT_S_14141_rep1_190729


title: LU13

description:
lung_ab_normalized_counts.csv
LU13


title: sort_seq__Fig2B_WT_G1_14141_rep3

description: Library name: Fig2B_WT_G1_14141_rep3_191014


title: LU14

description:
lung_ab_normalized_counts.csv
LU14


title: LU11

description:
lung_ab_normalized_counts.csv
LU11

value: IgG


title: sort_seq__Fig2B_WT_G1_14141_rep1

description: Library name: Fig2B_WT_G1_14141_rep1_190729


title: LIVER36

description:
liver_ab_normalized_counts.csv
LIV36

source_name: Liver

value: Liver

value: ZT 21


title: scRNA-seq_after_adjustment

city: Beijing

first: Qingya

last: Shi

address: 30 Shuangqing Road, Haidian District

department: Tsinghua Medicine

country: China

institute: Tsinghua University

description: Library name: After_adjustment

library_source: transcriptomic single cell

data_processing:
Raw sequencing data were processed using the 10x Genomics Space Ranger pipeline to produce FASTQ files.
The sequences were mapped to the hg38 genome for gene expression counting using Space Ranger's default parameters.
Assembly: hg38
Supplementary files format and content: barcodes.tsv.gz: list of spatial barcodes
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in Matrix Market Exchange Format

source_name: Peripheral blood mononuclear cells

extract_protocol:
Ten milliliters of peripheral venous blood were collected at 56- and 84-weeks post-transplant, followed by immediate PBMCs isolation using Ficoll-Hypaque density-gradient centrifugation. Viability exceeded 90%.
Chromium 5’ Library Construction: Fresh cells (300–600 viable cells/µL) were loaded into the Chromium system to form single-cell GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (53°C for 45 min, then 85°C for 5 min), and cDNA was amplified and quality-checked using an Agilent 4200. Libraries were constructed with a Single Cell 5′ Library and Gel Bead kit, sequenced with paired-end 150-bp reads on an Illumina NovaSeq6000.

value: Peripheral blood mononuclear cells


title: scRNA-seq_before_adjustment

description: Library name: Before_adjustment


title: LIVER35

description:
liver_ab_normalized_counts.csv
LIV35


title: LU10

description:
lung_ab_normalized_counts.csv
LU10


title: LIVER33

description:
liver_ab_normalized_counts.csv
LIV33


title: LIVER34

description:
liver_ab_normalized_counts.csv
LIV34


title: LU16

description:
lung_ab_normalized_counts.csv
LU16


title: LIVER32

description:
liver_ab_normalized_counts.csv
LIV32


title: scRNA-seq of Dorsal Forebrain, Ventral Forebrain, Dorsal Hindbrain, Ventral Hindbrain

city: Stanford

first: Arjun

last: Rajan

state: California

address: 265 Campus Drive

department: Developmental Biology

institute: Stanford University

description: Library name: AMXC_04

data_processing:
CellRanger v7 was used to performed read alginment to hg38 reference genome, barcode counting, and UMI counting
Scanpy was used to filter out low quality cells. Cells were annotated based on sample input. Data was normalized and log transformed.
Scanpy was used to generate UMAP projections following standard pipeline.
Assembly: hg38
Supplementary files format and content: .h5ad: h5ad file prepared using Scanpy containing annotated and QC'd cells from corresponding library

molecule: polyA RNA

source_name: cell culture

growth_protocol: D1 Definitive ectoderm media differentiation: D0 hPSCs were briefly washed before differentiation with DMEM/F12 to remove any traces of mTeSR Plus. They were then differentiated toward definitive ectoderm in CDM2 media supplemented with A8301 (1 µM, Tocris), Trametinib (250 nM, Cellagen Technology), C59 (1 µM, Tocris), and LDN193189 (100 nM, Stemgent) for 24 hours.  D2 Anterior neural ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 µM, Tocris), LDN193189 (100 nM, Stemgent), C59 (1 µM, Tocris), and Thiazovivin (1 µM, Tocris) for 24 hours.  Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells/cm2, and for experiments ending after more than 2 days of differentiation (i.e., towards a forebrain fate), 75,000 cells/cm2 were plated.  D2 Posterior neural ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 μM, Tocris), LDN193189 (100 nM, Stemgent), TTNPB (50 nM, Tocris), FGF2 (20 ng/mL, R&D Systems), XAV939 (1 μM, Tocris), and Thiazovivin (1 μM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells/cm2, and for experiments ending after more than 2 days of differentiation (i.e., toward a hindbrain fate), 150,000 cells/cm2 were plated.  D2 Border ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells at 37,500 cells/cm2 on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 µM, Tocris), PD0325901 (100 nM, Tocris), CHIR99012 (3 µM, Tocris), BMP4 (0.5 ng/mL, R&D Systems), and Thiazovivin (1 µM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging.  D2 Surface ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells at 37,500 cells/cm2 on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 µM, Tocris), PD0325901 (100 nM, Tocris), BMP4 (30 ng/mL, R&D Systems), C59 (1 µM, Tocris), and Thiazovivin (1 µM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging.  D4 Forebrain media differentiation: D2 anterior neural ectoderm cells were briefly washed with DMEM/F12 prior to addition of forebrain media. D3 forebrain media consisted of CDM2 supplemented with XAV939 (1 µM, Tocris), LDN193189 (100 nM, Stemgent), and FGF2 (20 ng/mL, R&D Systems), and was changed after 24 hours into D4 forebrain media, which consisted of CDM2 supplemented with XAV939 (1 µM) and LDN192189 (100 nM).  D4 Hindbrain media differentiation: D2 posterior neural ectoderm cells were briefly washed with DMEM/F12 prior to addition of hindbrain media. D3 and D4 hindbrain media consisted of CDM2 supplemented with CHIR99021 (0.75 µM, Tocris) and FGF2 (20 ng/mL, R&D Systems). Hindbrain media was replaced after 24 hours.  D7 Dorsal forebrain media differentiation: D4 forebrain progenitors were briefly washed with DMEM/F12 prior to addition of dorsal forebrain media. D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with CHIR99021 (2 µM, Tocris), Vismodegib (150 nM, Selleck Chemicals), and PD173074 (100 nM, Tocris). Dorsal forebrain differentiation was conducted for 72 hours, and dorsal forebrain media was replaced every 24 hours.  D7 Ventral forebrain media differentiation: D4 forebrain progenitors were briefly washed with DMEM/F12 prior to addition of ventral forebrain media. D5, D6, and D7 dorsal forebrain media consisted of CDM2 supplemented with XAV939 (1 µM, Tocris) and SAG 21K (5 nM, Tocris). Ventral forebrain differentiation was conducted for 72 hours, and ventral forebrain media was replaced every 24 hours.  D7 Dorsal hindbrain media differentiation: D4 hindbrain progenitors were briefly washed with DMEM/F12 prior to addition of dorsal hindbrain media. D5, D6, and D7 dorsal hindbrain media consisted of CDM2 supplemented with CHIR99021 (2 µM, Tocris), Vismodegib (150 nM, Selleck Chemicals), and LDN193189 (100 nM, Tocris). Dorsal hindbrain differentiation was conducted for 72 hours, and dorsal hindbrain media was replaced every 24 hours.  D7 Ventral hindbrain media differentiation: D4 hindbrain progenitors were briefly washed with DMEM/F12 prior to addition of ventral hindbrain media. D5, D6, and D7 ventral hindbrain media consisted of CDM2 supplemented with XAV939 (1 µM, Tocris) and SAG 21K (5 nM, Tocris). Ventral hindbrain differentiation was conducted for 72 hours, and ventral hindbrain media was replaced every 24 hours.  D14 Neuronal differentiation: hPSC-derived day 7 dorsal forebrain, day 7 ventral forebrain, day 7 dorsal hindbrain, and day 7 ventral forebrain progenitors were subsequently differentiated into neurons for 7 days. Neuronal differentiation media was modified from a previous report127. From day 8-14, cells were differentiated in BDNF (20 ng/ml, PeproTech), GDNF (20 ng/ml, PeproTech), Forskolin (10 μM, Tocris), 2-Phospho-L-ascorbic acid (200 μg/ml, Sigma-Aldrich), and DAPT (10 μM, R&D Systems), with different basal media used on each day of differentiation. The general approach was to gradually transition differentiating cells from CDM2 to Neurobasal differentiation media. Media composition was as follows: 1) D7-8 media (i.e., first 24 hours of neuronal differentiation) contained: 25% Neurobasal media (see below) + 75% CDM2 media was added; 2) D8-9 media (i.e., second 24 hours of neuronal differentiation): 50% Neurobasal media + 50% CDM2 media was added; 3) D9-10 (i.e., third 24 hours of neuronal differentiation), 75% Neurobasal media + 25% CDM2 media was added; 4) D10-14 (i.e., remaining 96 hours of neuronal differentiation), 100% Neurobasal media. In other words, throughout the entire neuronal differentiation phase, cells were exposed to the same concentration of neuronal differentiation signals (BDNF, GDNF, Forskolin, 2-Phospho-L-ascorbic acid, and DAPT) while a gradual change in basal media occurred. “Neurobasal media” is comprised of Neurobasal media (Thermo Fisher) supplemented with 1% v/v N2 (Thermo Fisher), 2% v/v B27 w/o Vitamin A (Thermo Fisher), and 1% v/v penicillin/streptomycin (Thermo Fisher). The media was passed through a sterilizing 0.22 μM filter before use.

extract_protocol:
hPSCs and differentiated ectoderm cells were enzymatically dissociated in Accutase (Thermo Fisher) at 37 ºC. Day 14 neuron-containing populations were dissociated in papain (Worthington). All samples were run through a 40 µM filter prior to fixation for 24 hours at 4C and storage at -80C.
Single cell gene expression was profiled using the Chromium Single Cell Gene Expression Flex kit (10x Genomics) according to the manufacturer’s recommendations. A multiplexed experimental design was used in which 10,000 cells were targeted to be recovered per probe barcode, and 5000 cells per sample were targeted to be captured on Chip Q. Library preparation was confirmed using Agilent Bioanalyzer (High Sensitivity DNA kit), and libraries were sequenced on a NovaSeq 6000.

value: cell culture

value: wild-type

value: DFB, VHB, DHB, VHB


title: scRNA-seq of Hindbrain

description: Library name: AMXC_03

value: HB


title: LIVER31

description:
liver_ab_normalized_counts.csv
LIV31


title: scRNA-seq of Forebrain

description: Library name: AMXC_02

value: FB


title: scRNA-seq of Dorsal Forebrain Neurons, Ventral Forebrain Neurons, Dorsal Hindbrain Neurons, Ventral Hindbrain Neurons

description: Library name: AMXC_05

value: DFB-N, VHB-N, DHB-N, VHB-N


title: LIVER30

description:
liver_ab_normalized_counts.csv
LIV30

value: depleted

value: ZT 17

value: Antibody depletion


title: LIVER29

description:
liver_ab_normalized_counts.csv
LIV29

value: WT


title: scRNA-seq of BE, SE

description: Library name: Sample5-6

value: BE, SE


title: scRNA-seq of hPSCs, DE, aNE, pNE

description: Library name: Sample1-4

value: hPSC, DE, aNE, pNE


title: pNE_H_RNA_2

description:
Library name: Posterior Neural Ectoderm to Hindbrain RNA-seq replicate 2
pNE-H_2

data_processing:
Bcl2Fastq (de-multiplexing performed by Novogene)
Read quality was assessed by FastQC
Index adaptors and low-quality base called were removed by TrimGalore
RNA-seq reads were aligned to hg38 genome using Kallisto
Gene-level counts were derived from transcript-level counts using tximport
Bigwig files were generated from Kallisto derived BAM files using deepTools
Assembly: Hg38
Supplementary files format and content: tpms.csv: Contains transcript per million (TPM) counts at the gene-level per sample
Supplementary files format and content: .bw: Track files per cell-type from merged replicates for hg38 genome to visualize transcript pileup in genome browser software

growth_protocol: D1 Definitive ectoderm media differentiation: D0 hPSCs were briefly washed before differentiation with DMEM/F12 to remove any traces of mTeSR Plus. They were then differentiated toward definitive ectoderm in CDM2 media supplemented with A8301 (1 µM, Tocris), Trametinib (250 nM, Cellagen Technology), C59 (1 µM, Tocris), and LDN193189 (100 nM, Stemgent) for 24 hours.  D2 Anterior neural ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 µM, Tocris), LDN193189 (100 nM, Stemgent), C59 (1 µM, Tocris), and Thiazovivin (1 µM, Tocris) for 24 hours.  Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells/cm2, and for experiments ending after more than 2 days of differentiation (i.e., towards a forebrain fate), 75,000 cells/cm2 were plated.  D2 Posterior neural ectoderm media differentiation: D1 definitive ectoderm cells were resplit using Accutase (Thermo Fisher) and plated as single cells on Geltrex-coated cell culture plates in CDM2 supplemented with A8301 (1 μM, Tocris), LDN193189 (100 nM, Stemgent), TTNPB (50 nM, Tocris), FGF2 (20 ng/mL, R&D Systems), XAV939 (1 μM, Tocris), and Thiazovivin (1 μM, Tocris) for 24 hours. Thiazovivin was added to promote cell survival after enzymatic passaging. For experiments ending after two days of differentiation, cells were plated at 37,500 cells/cm2, and for experiments ending after more than 2 days of differentiation (i.e., toward a hindbrain fate), 150,000 cells/cm2 were plated.  D4 Forebrain media differentiation: D2 anterior or posterior neural ectoderm cells were briefly washed with DMEM/F12 prior to addition of forebrain media. D3 forebrain media consisted of CDM2 supplemented with XAV939 (1 µM, Tocris), LDN193189 (100 nM, Stemgent), and FGF2 (20 ng/mL, R&D Systems), and was changed after 24 hours into D4 forebrain media, which consisted of CDM2 supplemented with XAV939 (1 µM) and LDN192189 (100 nM).  D4 Midbrain media differentiation: D2 anterior or posterior neural ectoderm cells were briefly washed with DMEM/F12 prior to addition of midbrain media. D3 midbrain media consisted of CDM2 supplemented with CHIR99021 (3 µM, Tocris) and was changed after 24 hours into D4 midbrain media, which consisted of CDM2 supplemented with CHIR99021 (3 µM) and FGF2 (20 ng/mL, R&D Systems).  D4 Hindbrain media differentiation: D2 anterior or posterior neural ectoderm cells were briefly washed with DMEM/F12 prior to addition of hindbrain media. D3 and D4 hindbrain media consisted of CDM2 supplemented with CHIR99021 (0.75 µM, Tocris) and FGF2 (20 ng/mL, R&D Systems). Hindbrain media was replaced after 24 hours.

extract_protocol:
Cells were lysed with Rneasy lysis buffer (Qiagen) and RNA was subsequently extracted using RNeasy Plus Kit (Qiagen)
Libraries were prepared by Novogene. Briefly: messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. Libraries were sequenced on a Novoseq 6000.

value: D1 DE media  -> D2 pNE media -> D4 Hindbrain Media


title: pNE_H_RNA_1

description:
Library name: Posterior Neural Ectoderm to Hindbrain RNA-seq replicate 1
pNE-H_1


title: pNE_M_RNA_2

description:
Library name: Posterior Neural Ectoderm to Midbrain RNA-seq replicate 2
pNE-M_2

value: D1 DE media  -> D2 pNE media -> D4 Midbrain Media


title: LU12

description:
lung_ab_normalized_counts.csv
LU12

value: Lung

value: ZT 1

value: IgG


title: sort_seq__Fig2B_WT_G1_14141_rep2

description: Library name: Fig2B_WT_G1_14141_rep2_190729

value: haploid

value: Mat A his3 leu2 met15 ura3


title: LIVER27

description:
liver_ab_normalized_counts.csv
LIV27

value: Liver


title: LU1

description:
lung_ab_normalized_counts.csv
LU1

value: ZT 5

value: anti Ly6G (1A8)


title: pNE_M_RNA_1

description:
Library name: Posterior Neural Ectoderm to Midbrain RNA-seq replicate 1
pNE-M_1

value: cell culture

value: wild-type


title: pNE_T_RNA_2

description:
Library name: Posterior Neural Ectoderm to Forebrain RNA-seq replicate 2
pNE-T_2

value: D1 DE media  -> D2 pNE media -> D4 Forebrain Media


title: LIVER25

description:
liver_ab_normalized_counts.csv
LIV25

value: ZT 17

value: Antibody depletion


title: pNE_T_RNA_1

description:
Library name: Posterior Neural Ectoderm to Forebrain RNA-seq replicate 1
pNE-T_1


title: LIVER24

description:
liver_ab_normalized_counts.csv
LIV24

value: depleted

value: ZT 13

value: WT


title: pNE_RNA_2

description:
Library name: Posterior Neural Ectoderm RNA-seq replicate 2
pNE_2

value: D1 DE media  -> D2 pNE media


title: LIVER23

description:
liver_ab_normalized_counts.csv
LIV23


title: pNE_RNA_1

description:
Library name: Posterior Neural Ectoderm RNA-seq replicate 1
pNE_1


title: LIVER22

description:
liver_ab_normalized_counts.csv
LIV22


title: aNE_H_RNA_2

description:
Library name: Anterior Neural Ectoderm to Hindbrain RNA-seq replicate 2
aNE-H_2

value: D1 DE media  -> D2 aNE media -> D4 Hindbrain media


title: LIVER21

description:
liver_ab_normalized_counts.csv
LIV21


title: aNE_H_RNA_1

description:
Library name: Anterior Neural Ectoderm to Hindbrain RNA-seq replicate 1
aNE-H_1


title: aNE_M_RNA_1

description:
Library name: Anterior Neural Ectoderm to Midbrain RNA-seq replicate 1
aNE-M_1

value: D1 DE media  -> D2 aNE media -> D4 Midbrain media


title: aNE_M_RNA_2

description:
Library name: Anterior Neural Ectoderm to Midbrain RNA-seq replicate 2
aNE-M_2


title: LIVER20

description:
liver_ab_normalized_counts.csv
LIV20

value: IgG


title: LIVER19

description:
liver_ab_normalized_counts.csv
LIV19


title: LIVER18

description:
liver_ab_normalized_counts.csv
LIV18

value: ZT 9


title: LIVER28

description:
liver_ab_normalized_counts.csv
LIV28


title: LIVER17

description:
liver_ab_normalized_counts.csv
LIV17


title: aNE_T_RNA_1

description:
Library name: Anterior Neural Ectoderm to Forebrain RNA-seq replicate 1
aNE-T_1

value: D1 DE media  -> D2 aNE media -> D4 Forebrain media


title: aNE_RNA_2

description:
Library name: Anterior Neural Ectoderm RNA-seq replicate 2
aNE_2

value: D1 DE media  -> D2 aNE media


title: LIVER14

description:
liver_ab_normalized_counts.csv
LIV14

value: Liver


title: LIVER26

description:
liver_ab_normalized_counts.csv
LIV26


title: hPSC_RNA_2

description:
Library name: hPSC RNA-seq replicate 2
hPSC_2

value: D0 hPSC media


title: LIVER12

description:
liver_ab_normalized_counts.csv
LIV12

value: ZT 5

value: anti Ly6G (1A8)


title: hPSC_RNA_1

description:
Library name: hPSC RNA-seq replicate 1
hPSC_1

value: cell culture

value: wild-type


title: pNE_H_ATAC_3

description: Library name: Posterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 3

data_processing:
Read quality for ATAC-seq data was checked using FastQC and size-distribution and transcriptional start site (TSS) enrichment was confirmed using ATAQV.
Reads were trimmed using Ngmerge
Trimmed reads were aligned to hg38 genome using bowtie2 --very-sensitive -I 10 -X 2000
Duplicate reads were removed using samblaster
Peaks were called on merged replicates using MACS2 and blacklisted regions were excluded using bedtools
TMM normalization was performed on each replicate using featureCounts and EdgeR.
Normalized bigwig files were generated for individual replicates using deeptools, and fragments larger than 120 bases were excluded, and bigwig file replicates were merged using wiggletools
Assembly: hg38
Supplementary files format and content: .bed: Peak files per cell-type from merged replicates called by MACS2 with blacklisted regions removed
Supplementary files format and content: .bw: z-score normalized track files per cell-type from merged replicates for hg38 genome with large fragments (>120bp) removed to visualize open chromatin regions   in genome browser software

extract_protocol:
Nuceli were extracted using a modified version of the OmniATAC-seq protocol. 50,000 live, freshly isolated cells were washed with 50 µl cold 1X PBS and pelleted. 50 µl of cold lysis buffer (0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v Digitonin in resuspension buffer, comprised of 10 mM Tris-HCL, 10 mM NaCl, and 3 mM MgCl2) was added to each sample and incubated for 3 minutes on ice. 1 mL of wash buffer (resuspension buffer with 0.1% v/v Tween-20) was added to each sample before centrifugation at 500xg for 10 minutes at 4C. The supernatant was discarded, and each nuclei pellet was resuspended in 50 µl of transposition buffer (25 µl 2X TD Buffer, 16.5 µl 1X PBS, 0.5 µl 10% Tween-20, 0.5 µl 1% Digitonin, 2.5 µl Tn5 Transposase, 5 µl nuclease-free water). The mixture was incubated for 30 minutes at 37°C on a thermomixer set to 1000 rpm. Transposed DNA was isolated using the Qiagen MinElute Reaction Cleanup Kit, as per manufacturer’s instructions.
Libraries were generated using Illumina Nextera DNA Unique Dual Indexes and NEBNext High-Fidelity 2X PCR Master Mix. Libraries were double-side purified using AMPure XP beads, and quality was assessed using Agilent Bioanalyzer (High Sensitivity DNA kit). Sequencing was conducted on a NovaSeq 6000.

value: D1 DE media  -> D2 pNE media -> D4 Hindbrain Media


title: pNE_H_ATAC_2

description: Library name: Posterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 2


title: LIVER11

description:
liver_ab_normalized_counts.csv
LIV11

value: depleted

value: Antibody depletion


title: LIVER09

description:
liver_ab_normalized_counts.csv
LIV09

value: WT


title: LIVER10

description:
liver_ab_normalized_counts.csv
LIV10


title: pNE_H_ATAC_1

description: Library name: Posterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 1


title: pNE_M_ATAC_3

description: Library name: Posterior Neural Ectoderm to  Midbrain ATAC-seq replicate 3

value: D1 DE media  -> D2 pNE media -> D4 Midbrain Media


title: pNE_M_ATAC_1

description: Library name: Posterior Neural Ectoderm to  Midbrain ATAC-seq replicate 1


title: aNE_T_RNA_2

description:
Library name: Anterior Neural Ectoderm to Forebrain RNA-seq replicate 2
aNE-T_2


title: LIVER08

description:
liver_ab_normalized_counts.csv
LIV08


title: LIVER16

description:
liver_ab_normalized_counts.csv
LIV16


title: LIVER06

description:
liver_ab_normalized_counts.csv
LIV06

value: ZT 1


title: LIVER07

description:
liver_ab_normalized_counts.csv
LIV07


title: pNE_M_ATAC_2

description: Library name: Posterior Neural Ectoderm to  Midbrain ATAC-seq replicate 2


title: pNE_T_ATAC_3

description: Library name: Posterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 3

value: D1 DE media  -> D2 pNE media -> D4 Telencephalon Media


title: aNE_RNA_1

description:
Library name: Anterior Neural Ectoderm RNA-seq replicate 1
aNE_1


title: LIVER15

description:
liver_ab_normalized_counts.csv
LIV15

value: IgG


title: LIVER04

description:
liver_ab_normalized_counts.csv
LIV04


title: LIVER03

description:
liver_ab_normalized_counts.csv
LIV03


title: pNE_T_ATAC_1

description: Library name: Posterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 1


title: aNE_H_ATAC_1

description: Library name: Anterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 1

value: D1 DE media  -> D2 aNE media -> D4 Hindbrain media


title: LIVER05

description:
liver_ab_normalized_counts.csv
LIV05


title: LIVER01

description:
liver_ab_normalized_counts.csv
LIV01


title: LIVER02

description:
liver_ab_normalized_counts.csv
LIV02


title: LIVER13

description:
liver_ab_normalized_counts.csv
LIV13

value: ZT 9


title: pNE_T_ATAC_2

description: Library name: Posterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 2


title: aNE_H_ATAC_2

description: Library name: Anterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 2


title: aNE_H_ATAC_3

description: Library name: Anterior Neural Ectoderm to  Hindbrain ATAC-seq replicate 3


title: aNE_M_ATAC_2

description: Library name: Anterior Neural Ectoderm to  Midbrain ATAC-seq replicate 2

value: D1 DE media  -> D2 aNE media -> D4 Midbrain media


title: aNE_M_ATAC_1

description: Library name: Anterior Neural Ectoderm to  Midbrain ATAC-seq replicate 1


title: aNE_T_ATAC_3

description: Library name: Anterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 3

value: D1 DE media  -> D2 aNE media -> D4 Telencephalon media


title: zt9_co1_INT13

description:
gut_ab_normalized_counts.csv
zt9_co1_INT13

source_name: Gut

value: Gut


title: zt9_co3_INT15

description:
gut_ab_normalized_counts.csv
zt9_co3_INT15


title: zt9_1a8_3_INT18

description:
gut_ab_normalized_counts.csv
zt9_1a8_3_INT18


title: aNE_M_ATAC_3

description: Library name: Anterior Neural Ectoderm to  Midbrain ATAC-seq replicate 3


title: zt9_1a8_2_INT17

description:
gut_ab_normalized_counts.csv
zt9_1a8_2_INT17


title: pNE_ATAC_2

description: Library name: Posterior Neural Ectoderm ATAC-seq replicate 2

value: D1 DE media  -> D2 pNE media


title: aNE_T_ATAC_2

description: Library name: Anterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 2


title: zt5_1a83bis_INT12

description:
gut_ab_normalized_counts.csv
zt5_1a83bis_INT12

value: ZT 5

value: anti Ly6G (1A8)


title: aNE_T_ATAC_1

description: Library name: Anterior Neural Ectoderm to  Telencephalon ATAC-seq replicate 1

value: cell culture

value: wild-type


title: zt5_co1_INT7

description:
gut_ab_normalized_counts.csv
zt5_co1_INT7


title: zt5_co3_INT9

description:
gut_ab_normalized_counts.csv
zt5_co3_INT9


title: zt5_co2_INT8

description:
gut_ab_normalized_counts.csv
zt5_co2_INT8


title: zt9_1a8_1_INT16

description:
gut_ab_normalized_counts.csv
zt9_1a8_1_INT16

value: depleted


title: pNE_ATAC_1

description: Library name: Posterior Neural Ectoderm ATAC-seq replicate 1


title: aNE_ATAC_1

description: Library name: Anterior Neural Ectoderm ATAC-seq replicate 1

value: D1 DE media  -> D2 aNE media


title: zt5_1a82_INT11

description:
gut_ab_normalized_counts.csv
zt5_1a82_INT11

value: Antibody depletion

value: WT


title: zt5_1a81_INT10

description:
gut_ab_normalized_counts.csv
zt5_1a81_INT10


title: pNE_ATAC_3

description: Library name: Posterior Neural Ectoderm ATAC-seq replicate 3


title: DE_ATAC_3

description: Library name: Definitive Ectoderm ATAC-seq replicate 3

value: D1 DE media


title: aNE_ATAC_2

description: Library name: Anterior Neural Ectoderm ATAC-seq replicate 2


title: zt9_co_2_INT14

description:
gut_ab_normalized_counts.csv
zt9_co_2_INT14


title: zt21_bm1_INT31

description:
gut_ab_normalized_counts.csv
zt21_bm1_INT31

value: ZT 21


title: zt21_1a83_INT36

description:
gut_ab_normalized_counts.csv
zt21_1a83_INT36


title: hPSC_ATAC_3

description: Library name: hPSC ATAC-seq replicate 3

value: D0 hPSC media


title: zt21_1a82_INT35

description:
gut_ab_normalized_counts.csv
zt21_1a82_INT35


title: hPSC_ATAC_2

description: Library name: hPSC ATAC-seq replicate 2


title: zt21_bm3_INT33

description:
gut_ab_normalized_counts.csv
zt21_bm3_INT33

value: IgG


title: zt17_co3_INT27

description:
gut_ab_normalized_counts.csv
zt17_co3_INT27

value: ZT 17


title: hPSC_ATAC_1

description: Library name: hPSC ATAC-seq replicate 1


title: DE_ATAC_2

description: Library name: Definitive Ectoderm ATAC-seq replicate 2


title: DE_ATAC_1

description: Library name: Definitive Ectoderm ATAC-seq replicate 1


title: zt21_bm2_INT32

description:
gut_ab_normalized_counts.csv
zt21_bm2_INT32


title: aNE_ATAC_3

description: Library name: Anterior Neural Ectoderm ATAC-seq replicate 3


title: overTST_CTR4

city: Fribourg

first: Karim

last: Zuhra

address: Chemin du Musée, 18

country: Switzerland

institute: University of Fribourg

description: Library name: Sample12

data_processing:
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Below are the statistics of mapping the reads to the reference genome. Unique gene hit counts.txt were calculated by using featurecounts.txt from the Subread package v.1.5.2. The hit counts.txt were summarized and reported using the gene_id feature in the annotation file. Only unique reads that fell within exon regions were counted. If a strand-specific library preparation was performed, the reads were strand-specifically counted.
Assembly: GRCh38
Supplementary files format and content: txt files with unique gene hit counts.txt.

extract_protocol:
Cells were washed twice and RNA extraction and purification were performed using the NucleoSpin® RNA plus kit (Macherey-Nagel, 740984.250) according to the manufacturer’s instructions. RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific) by measuring the absorbance at 260/280 nm ratio.
Library construction was performed by Genewiz (Azenta Life Sciences).

value: Hepatocarcinoma cell

value: overTST


title: zt17_co1_INT25

description:
gut_ab_normalized_counts.csv
zt17_co1_INT25

value: Gut


title: 59_Rotenone_EC50_96h_Eppi-89_Index-D11

city: Leipzig

first: Paul

last: Michaelis

address: Permoserstr. 15

institute: Helmholtz-Centre for Environmental Research (UFZ)

data_processing:
alignment to GRCz11 with HISAT2 (v.2.1.0)
filter reads by quality > 30 (Samtools view v.1.15.1)
generate counts with featureCounts (v.2.0.3)
QC with MultiQC (v.1.11)
Assembly: GRCz11
Supplementary files format and content: counts.tsv: unnormalized counts
Supplementary files format and content: sample_info.tsv: additional metadata for samples

organism: Danio rerio

source_name: whole body

growth_protocol: Wild-type adult zebrafish (D. rerio, strain OBI/WIK, generation F3) were kept at 26°C in 20 L aquaria with 1-2 fish/L, fed twice a day and on a 14h:10h light dark cycle.

extract_protocol:
Trizol, homogenization by a FastPrep 50 (MP Biomedicals), followed by Total RNA Extraction Kit MagMAX, RIN>7
NEBnext Ultra II stranded RNA library preparation kit for Illumina (NEB, Beverly, MA, USA)

treatment_protocol: Exposure to Sorafenib or Rotenone at 2 or 24 hpf, 20 embryos per replicate, incubation at 26°C with 12:12 light:dark photoperiod

value: whole body

value: treatment

tag: substance

value: Rotenone

tag: concentration

tag: unit

value: nM

tag: exposure start_hpf

tag: time hpf


title: zt17_co2_INT26

description:
gut_ab_normalized_counts.csv
zt17_co2_INT26


title: overTST_CTR1

description: Library name: Sample9


title: shTST_CTR3

description: Library name: Sample7

value: shTST


title: overTST_CTR3

description: Library name: Sample11


title: overTST_CTR2

description: Library name: Sample10


title: 57_Rotenone_EC50_96h_Eppi-87_Index-B11


title: shTST_CTR2

description: Library name: Sample6


title: zt17_1a83_INT30

description:
gut_ab_normalized_counts.csv
zt17_1a83_INT30

value: depleted

value: anti Ly6G (1A8)


title: shTST_CTR4

description: Library name: Sample8


title: 58_Rotenone_EC50_96h_Eppi-88_Index-C11


title: zt17_1a82_INT29

description:
gut_ab_normalized_counts.csv
zt17_1a82_INT29

value: Antibody depletion


title: 42_Rotenone_EC50_48h_Eppi-64_Index-C9

value: WT


title: zt17_1a81_INT28

description:
gut_ab_normalized_counts.csv
zt17_1a81_INT28


title: WT_CTR4

description: Library name: Sample4


title: zt21_1a81_INT34

description:
gut_ab_normalized_counts.csv
zt21_1a81_INT34


title: WT_CTR3

description: Library name: Sample3


title: zt13_co3_INT21

description:
gut_ab_normalized_counts.csv
zt13_co3_INT21

value: ZT 13


title: WT_CTR2

description: Library name: Sample2


title: 41_Rotenone_EC50_48h_Eppi-63_Index-B9


title: WT_CTR1

description: Library name: Sample1


title: zt13_co2_INT20

description:
gut_ab_normalized_counts.csv
zt13_co2_INT20


title: 40_Rotenone_EC50_48h_Eppi-61_Index-A9


title: zt13_co1_INT19

description:
gut_ab_normalized_counts.csv
zt13_co1_INT19


title: Gly2

description: Library name: Sample5

value: Glycine 10 mM


title: Gly3

description: Library name: Sample6


title: Dgal-3

city: Shanghai

first: Luze

address: Xinsongjiang Road 650

institute: Shanghai General Hospital

description: Library name: Sample6

data_processing:
. The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version:2.0.4)
Assembly: Sequencing Technology
Supplementary files format and content: Tab-separated values files and matrix files

source_name: skeletal muscle

extract_protocol:
Total RNA (2μg) was extracted from N0R-10 followed by library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® ).
The protocol of high-throughput sequencing was fully according to the manufacturer's instructions (Illumina)

value: skeletal muscle

value: skeletal muscle fibroblast

value: D-gal


title: 28_Rotenone_EC50_36h_Eppi-40_Index-D7


title: zt13_1a8_3_INT24

description:
gut_ab_normalized_counts.csv
zt13_1a8_3_INT24


title: Gly1

description: Library name: Sample4

value: Hepatocarcinoma cell


title: Dgal-2

description: Library name: Sample5


title: zt13_1a8_2_INT23

description:
gut_ab_normalized_counts.csv
zt13_1a8_2_INT23


title: 27_Rotenone_EC50_36h_Eppi-38_Index-C7


title: CTR3

description: Library name: Sample3

value: Vehicle


title: shTST_CTR1

description: Library name: Sample5


title: 26_Rotenone_EC50_36h_Eppi-37_Index-B7


title: Con-3

description: Library name: Sample3

value: PBS


title: CTR2

description: Library name: Sample2


title: 60_Rotenone_LC10_48h_Eppi-50_Index-E11

value: whole body


title: CTR1

description: Library name: Sample1


title: zt13_1a8_1_INT22

description:
gut_ab_normalized_counts.csv
zt13_1a8_1_INT22

value: Gut


title: zt1_co3_INT3

description:
gut_ab_normalized_counts.csv
zt1_co3_INT3

value: ZT 1

value: IgG


title: Con-2

description: Library name: Sample2


title: 50_Rotenone_LC10_96h_Eppi-74_Index-C10

value: treatment

value: Rotenone

value: nM


title: Con-1

description: Library name: Sample1


title: zt1_co2_INT2

description:
gut_ab_normalized_counts.csv
zt1_co2_INT2

value: Antibody depletion


title: 49_Rotenone_LC10_96h_Eppi-72_Index-B10

value: WT


title: zt1_co1_INT1

description:
gut_ab_normalized_counts.csv
zt1_co1_INT1


title: 48_Rotenone_LC10_96h_Eppi-71_Index-A10


title: MKC177, PGK (EPOR) 2

city: San Francisco

first: Prathamesh

last: Chati

state: CA

address: 1975 4th Street

institute: University of California, San Francisco

description:
Library name: MKC177
Column name in both processed files: MKC177

data_processing:
Quality control performed by Novogene.
FASTQ files were aligned to the GRCh38 reference genome extended with the synEPOR target sequence and quantified using Salmon (v1.9.0) with default parameters.
Outputted NumReads and TPM for each sample were aggregated into two separate files and were used for downstream analyses.
Assembly: GRCh38 extended with synEPOR sequences.
Supplementary files format and content: CSV files contain sample names along the columns and gene names (symbols) along rows.

source_name: Peripheral blood

growth_protocol: Human CD34+ HSPCs were cultured in StemSpan SFEMII or Good Manufacturing Practice Stem Cell Growth Medium supplemented with the following cytokines: stem cell factor (100 ng/mL), thrombopoietin (100 ng/mL), Fms-like tyrosine kinase 3 ligand (100 ng/mL), interleukin-6 (100 ng/mL), as well as streptomycin (20 mg/mL) and penicillin (20 U/mL). Additionally, 35 nM of UM171 was added. Cells were maintained in a humidified incubator at 37°C, 5% CO2, and 5% O2.

extract_protocol:
RNA Extraction: Total RNA was extracted from cell pellets (approximately 1×10⁶ cells per sample) using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. RNA quality and quantity were assessed prior to sequencing library preparation.
Library Construction: RNA sequencing libraries were prepared using the SMARTer mRNA-Seq Library Prep Kit following the manufacturer's instructions. Approximately 1.3 μg of total RNA was used for library preparation. The libraries were validated for quality and quantified prior to sequencing.

treatment_protocol: Adipogenic differentiation: Following genome editing, human CD34+ HSPCs were cultured in a three-phase differentiation system. During Phase 1 (Day 0–7), cells were cultured at 1×10⁵ cells/mL in SFEMII medium supplemented with stem cell factor (10 ng/mL), interleukin-3 (1 ng/mL), and transferrin (200 μg/mL). During Phase 2 (Day 7–10), interleukin-3 was removed, and transferrin concentration was maintained. During Phase 3 (Day 11–14), transferrin concentration was increased to 1 mg/mL. Differentiation conditions included either the addition of recombinant EPO (3 U/mL) or the absence of EPO with the addition of 1 nM BB dimerizer.

value: Peripheral blood

value: RBC

value: PGK (synEPOR)

value: Erythroid differentiation

value: Day 14


title: DS_177_1, PGK (EPOR) 1

description:
Library name: DS_177_1
Column name in both processed files: DS_177_1


title: Naïve MHCIIhi CD11c+ B220- cells, GX + ADT

city: Newark

first: Yosuke

last: Kumamoto

state: NJ

address: 205 South Orange Ave

department: Center for Immunity and Inflammation

institute: Rutgers New Jersey Medical School

description:
polyA RNA + protein
GX (3' mRNA) library + ADT: R1 contains cell barcode and UMI. R2 contains transcript and antibody-derived tag reads.

library_source: other

data_processing:
Cell Ranger (v3.0.1) and Loupe browser were used for data analysis
Further data processing, analysis and visualisation performed using Partek Flow (Partek Inc)
Assembly: mm10
Supplementary files format and content: Output Files: Naive_GX_processed_data.txt, PapOVA_d1_GX_processed_data.txt
Supplementary files format and content: Output Files: Naive_ADT_processed_data.txt, PapOVA_d1_ADT_processed_data.txt
Library strategy: CITE-seq

molecule: other

source_name: Skin draining lymph node

extract_protocol:
MHCIIhi CD11c+ B220- cells were sorted from popliteal lymph nodes of immunized and naïve mice and used to generate Gel Beads-in-Emulsion (GEMs) using the Chromium Single-Cell 3’ Reagent v3 kit (1000092, 10X Genomics) according to the manufacturer’s protocol.
Cell suspensions with a >90% viability was mixed with RT reagents and loaded onto Chromium Chip B, PN-1000074 along with Gel beads and Partitioning oil in the recommended order and then the chip was processed through 10X Chromium Controller for the generation of GEMs, followed by RT, Cleanup, and cDNA amplification. After the cDNA amplification, size selection was used to separate the cDNA molecules for 3’ Gexp (Gene Expression) library and ADT library construction

value: Skin draining lymph node

value: MHCIIhi CD11c+ B220-

value: unimmunized

tag: library type

value: mRNA + ADT

tag: adt

value: TotalSeq-A0566 anti-mouse CD301b (MGL2), TotalSeq-A0106 anti-mouse CD11c, TotalSeq-A0014 anti-mouse/human CD11b, TotalSeq-A0212 anti-mouse CD24, TotalSeq-A0002 anti-mouse CD8a, TotalSeq-A0422 anti-mouse CD172a (SIRPa), TotalSeq-A0568 anti-mouse/rat XCR1, TotalSeq-A0117 anti-mouse I-A/I-E, TotalSeq-A0202 anti-mouse CD64 (FcɣRI), TotalSeq-A0013 anti-mouse Ly-6c, TotalSeq-A0201 anti-mouse CD103, TotalSeq-A0449 anti-mouse CD326 (Ep-CAM)


title: DS_177_3, PGK (EPOR) 3

description:
Library name: DS_177_3
Column name in both processed files: DS_177_3

tag: cell line


title: zt1_1a83_INT6

description:
gut_ab_normalized_counts.csv
zt1_1a83_INT6

value: Gut

value: depleted

value: ZT 1

value: anti Ly6G (1A8)


title: 33_Rotenone_LC10_48h_Eppi-49_Index-A8

value: whole body

value: treatment

value: Rotenone

value: nM


title: 2209_FB

description:
Hashtag antibody
raw_10x_data.rds

data_processing:
Fastq files were generated using 'bcl2fastq' conversion software (version 2.20) from Illumina with specific sample indexes corresponding to each sample
Resulting data was then imported and processed into Seurat object using Seurat R package
Assembly: mm10
Supplementary files format and content: processed counts and ADT in seurat object: raw_10x_data.rds
Library strategy: CITE-seq

molecule: protein

source_name: Neutrophils (CD45+, Cd11b+, Ly6g+), other leukocytes (Cd45+, Ly6g-) and structural cells (Cd31+, Epcam+, mEF-SK4+)

extract_protocol:
Back skin was harvested and processed to obtain single-cell suspensions. Samples were barcoded with Oligo-tagged hashtag antibodies (Biolegend) and incubated with an antibody cocktail of Fixable viability dye eFluor780 (eBioscience), CD45-BUV395, CD11b-BV650, Ly6G-PE, EPCAM-APC, mFESK-4-APC, and CD31-APC. Live neutrophils (CD45+CD11b+Ly6G+), other immune cells (CD45+Ly6G-), and structural cells (CD45-Dump+) were isolated by FACS-based sorting.
Library preparation (Chromium Next GEM Single Cell 3’ GEM v3, 10X Genomics) and the subsequent sequencing (Novaseq 6000 instrument, Illumina) were performed by the Yale Center for Genome Analysis (YCGA), according to the manufacturer’s instructions (10X Genomics).

tag: cell type

value: mixed sample (depleted, control)

value: HTO

tag: hto

value: TotalSeq™-B0305 anti-mouse Hashtag 5 Antibody (depleted), TotalSeq™-B0306 anti-mouse Hashtag 6 Antibody (control)


title: DS_176_3, EPOR 3

description:
Library name: DS_176_3
Column name in both processed files: DS_176_3

tag: tissue

value: Peripheral blood

value: RBC

tag: genotype

value: EPOR (synEPOR)

value: Erythroid differentiation

value: Day 14


title: zt1_1a82_INT5

description:
gut_ab_normalized_counts.csv
zt1_1a82_INT5

value: Antibody depletion

tag: treatment

value: WT


title: Dgal-1

description: Library name: Sample4

value: skeletal muscle

value: skeletal muscle fibroblast

value: D-gal


title: 32_Rotenone_LC10_48h_Eppi-47_Index-H7

value: whole body


title: 2209_scRNA

description:
polyA RNA
raw_10x_data.rds

value: Neutrophils (CD45+, Cd11b+, Ly6g+), other leukocytes (Cd45+, Ly6g-) and structural cells (Cd31+, Epcam+, mEF-SK4+)

value: mRNA


title: zt1_1a81_INT4

description:
gut_ab_normalized_counts.csv
zt1_1a81_INT4

value: Gut

value: depleted

value: ZT 1

value: anti Ly6G (1A8)

tag: strain


title: 18_Rotenone_LC10_36h_Eppi-24_Index-B6

value: treatment

value: Rotenone

value: nM