title: Liver_DSS_Inulin13C_RNA_rep3

city: Philadelphia

first: Peder

middle: J

last: Lund

state: PA

address: 3400 Civic Center Blvd, Smilow 9-176

institute: University of Pennsylvania

description: processed data file: results_deseq2_liver.csv

data_processing:
Paired-end sequencing data (38 and 37 cycles for read 1 and read 2 with 6 cycle index) was acquired on an Illumina NextSeq 500 instrument.  FASTQ generation was provided by BaseSpace (Illumina) with GenerateFASTQWorker with bcl2fastq2.
Sequencing reads were aligned to the mouse mm10 genome assembly or the mm10 genome concatenated with the Drosophila dm6 genome in the case of the ChIP with the exogenous spike-in, using STAR (v2.5.2a).  For ChIP-seq, the following parameters were used: --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --alignMatesGapMax 1000000.  For RNA-seq, the following parameters were used: --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMax 1000000.
Sam files were filtered for unique alignments (MAPQ = 255) before conversion to bam and then bed files.  Read pairs were condensed into a single bed entry allowing for a maximum fragment length of 1000 bp.  Read pairs matching to different chromosomes were discarded.  Bed files for RNA-seq were produced with the -split option of bedtools enabled to eliminate introns.  For track visualization, RNA-seq data was normalized to total read counts (RPM) and read pairs were left unpaired.  Software tools included samtools (v1.1), Python (v3.6.3), and bedtools (2.15.0).
RNA-seq data was analyzed quantitatively with HTSeq (v0.10.0, parameters -f sam -r name -s reverse -t exon -i gene_id --nonunique none --secondary-alignments ignore --supplementary alignments ignore) and DESeq2.  For the RNA-seq data from the DSS colitis study, the pooled library was sequenced once and then a second pool was created and sequenced to obtain adequate sequencing depth across all samples.  After alignment, sam files from the two sequencing runs were concatenated and filtered for uniquely mapping reads for quantitative analysis by HTSeq and DESeq2.  Within a given tissue and disease state (e.g. healthy cecum), samples from the Un and 13C inulin groups (n = 3 each) were treated as replicates (n = 6) since the isotope label did not have a strong effect on gene expression.  Liver samples originated from different mice than the cecum and colon samples (i.e. cecum and colon but not liver represent paired samples).  Cecum and colon data were loaded into DESeq2 separately from liver data.  Log2 ratios of DSS/H2O were calculated separately for each tissue.
For ChIP-seq data, normalization to the exogenous reference chromatin in lieu of sequencing depth was performed as described previously (Fursova et al, 2019, Mol. Cell).  Briefly, the number of fragments matching to the mm10 or dm6 genomes were determined from uniquely aligning read pairs.  These numbers were used to calculate downsampling factors, corrected for any variations in reference chromatin abundance in the corresponding input samples, with the largest factor being equal to 1.  Sam files containing unique alignments for the H4ac4 ChIP samples were then downsampled according to these factors such that equivalent numbers of Drosophila reads were obtained across all samples.  Downsampled sam files were then converted to bam and bed files.  The mm10 and dm6 fragments were allocated to separate bed files for generating bigWig files, now calibrated to the reference chromatin, with an in-house script.  DeepTools was used for additional analysis and visualization.
Genome_build: mm10 or mm10 concatenated with dm6
Supplementary_files_format_and_content: Csv files contain quantitative information on RNA-seq data as analyzed by DESEQ2.  The fold-change indicates log2(GF/Conv) or log2(DSS/H2O).

source_name: Frozen liver tissue

extract_protocol:
Total RNA was extracted from aliquots of frozen tissues using the RNeasy kit (Qiagen).  Homogenization was carried out in the provided lysis buffer using bead beating (Precellys homogenizer, Bertin Technologies).
The NEBNext Ultra or Ultra II Directional RNA Library Prep Kit for Illumina with the mRNA magnetic isolation module (New England Biolabs) was used to prepare sequencing libraries from 1 µg of total RNA.

treatment_protocol: Mice (C57BL/6, female, 8-10 weeks, Jackson Labs) were acclimated to the AIN76a diet over the preceding 3 days and then had colitis induced with 2% dextran sodium sulfate (MP Bio, 36-50 kDa) in the drinking water on day 0. On days 6 and 7, mice received an oral gavage of 100 mg of U-13C-labeled or unlabeled inulin (IsoLife) dissolved in 200 µl PBS.  On day 8, mice were sacrificed by cervical dislocation after a 4 hour fast. Livers, colons, ceca, and cecal contents were harvested and flash frozen in liquid nitrogen.

value: DSS colitis mice with 13C-labeled inulin

tag: tissue/cell type

value: Frozen liver tissue


title: Monkey_GastrocnemiusTendon_Female_1503

status: Public on Dec 12 2022

type: SRA

city: North Chicago

first: Terry

last: Van Vleet

state: IL

address: 1 N Waukegan Rd

country: USA

institute: AbbVie

description:
Monkey_GastrocnemiusTendon_Female_1503
Cyno_FPKM_table_Mar22.xlsx

library_source: transcriptomic

data_processing:
ArrayStudio
Sequence reads were trimmed for adaptor sequence/low-quality sequence using ArrayStudio
Trimmed sequence reads were mapped to Mouse.B38, Rat.B6.0, Dog.CanFam3.1, and Macaca_fascicularis_5.0
Read count extraction and normalization were performed using ArrayStudio
Assembly: Mouse.B38, Rat.B6.0, Dog.CanFam3.1, and Macaca_fascicularis_5.0
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

molecule: total RNA

organism: Macaca fascicularis

source_name: Tendons

extract_protocol:
RNA was harvested using Rneasy mini kit (Qiagen). 200-300ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using TruSeq Stranded Total RNA Library Prep Kit Gold following manufacturer's protocols.

treatment_protocol:
NA
NA

tag: tissue

value: Tendons

tag: Sex

value: Female

tag: treatment

value: untreated


title: Liver_DSS_Inulin13C_RNA_rep2

organism: Mus musculus

tag: condition


title: MCF-7 CapQuant-seq 1

city: New York

first: Vladimir

last: Despic

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

data_processing:
Removal of low quality reads and 3'-end adapter trimming was performed with flexbar v2.5.
Removal of duplicated reads was accomplished with pyFastqDuplicateRemover.py within the pyCRAC package v1.3.2.
Read trimming was performed using seqtk package v1.3.
Read alignement to fly, mouse or human genome was conducted using Bowtie v1.2.3.
5'-end read coverage was calculated per each genomic position using BEDTools v2.28.0.
Genome_build: Drosohila melanogaster; dm6, Mus musculus; mm10, Homo sapiens; hg38.

molecule: polyA RNA

organism: Homo sapiens

source_name: cell culture

growth_protocol: MCF-7 cells were maintained in DMEM supplemented with 10% FBS, 100 U Penicillin-Streptomycin at 37°C and 5% CO2.

extract_protocol:
CapTag-seq
Total RNA was extracted using TRIzol reagent according to the manufacturers instructions followed by isopropanol precipitation.  Poly(A)+ RNA was isolated from total RNA using magnetic oligo(dT)25 beads (NEB).

tag: cell line/tissue

tag: genotype

value: wild type

tag: purification

value: oligo(dT)25 beads


title: WT ActD-TSS-seq _0 hrs 1

description:
WT_halflife-rep1.xlsx
HEK293T-TSN-list.xlsx

data_processing:
library strategy: TSS-seq
Low quality reads were removed and the Illumina small RNA-seq 3' end adapter (DA3; 5'-TGGAATTCTCGGGTGCCAAGG-3') was trimmed off the high quality  sequencing reads using flexbar v2.5: flexbar -r sample.fastq -f i1.8 -a DA3.fasta --pre-trim-phred 30 -n 20 -s -t output --min-read-length 18. Duplicated reads were then removed using pyFastqDuplicateRemover.py script. Next, only the forawrd (R1) sequencing reads were considered for further analysis. First, a 8-nucleotide-long UMI was removed from the sequencing read 5' end, followed by read trimming to 25 bp length using seqtk. The trimmed reads were first mapped to dm6 genome build with bowtie using the following parameters: bowtie -f -v 1 -m 8 --best --strata -S -p 10 dm6.genome sample.fasta --un unmapped.fasta dm6.sam. The remaining unmapped reads were then mapped to hg38 genome build with bowtie using the following parameters: bowtie -f -v 1 -m 8 --best --strata -S -p 10 hg38.genome.index unmapped.fasta hg38.sam. Half-lives per TSN isoform were calculated by first-order decay kinetics.
Genome_build: Human: hg38; Fly: dm6
Supplementary_files_format_and_content: Text file contains measured mRNA half-lives in two independent samples from CMTR2 WT and KO cells. Additionally, the file contains differences in average half-lives between CMTR2 KO and WT samples.

growth_protocol: HEK293T cells were maintained in DMEM supplemented with 10% FBS, 100 U Penicillin-Streptomycin at 37°C and 5% CO2.

extract_protocol:
Total RNA was extracted with TRIzol reagent and isopropanol precipitation. Poly(A)+ RNA was extracted using oligo(dT)25 megnetic beads.
Libraries were prepared using modified version of the TSS-seq protocol.

treatment_protocol: Cells were treated with 5 µg ml-1 actinomycin D for 0 h.

tag: cell line

value: ActD, 0 hrs


title: A549 CapQuant-seq 2

growth_protocol: A549 cell were maintained in Ham's F-12K medium supplemented with 10% FBS, 100 U Penicillin-Streptomycin at 37°C and 5% CO2.


title: A549 CapQuant-seq 1


title: Monkey_GastrocnemiusTendon_Female_1501

description:
Monkey_GastrocnemiusTendon_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Monkey_Gallbladder_Female_1503

description:
Monkey_Gallbladder_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Gallbladder

value: Gallbladder


title: Myeloid cells from naïve humanized mice, hashtag library

city: Bar Harbor

first: Guangwen

last: Ren

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

description: Hashtag-derived oligonucleotide (HTO)

data_processing:
The raw data (FASTQ file) of each library were produced and demultiplexed using Illumina bcl2fastq 2.20.0.422.
For all libraries, R1 contains a 16bp cell barcode + 12bp UMI barcode and R2 contains the biological reads. If present, I1 and I2 contains the index which is needed for 10X CellRanger pipelines.
An unfiltered digital gene expression matrix was generated for each gene expression library against the 10X Genomics reference genome (GRCh38-2020-A) build using CellRanger v6.1.1
A digital counts matrix was generated for each hashtag library using CITE-Seq-Count. This produces a matrix of counts with hashtag labels for rows and cell barcodes for columns.
Assembly: GRCh38-2020-A
Supplementary files format and content: Transcriptomic and HashTag (HTO) counts matrices in Matrix Market sparse CSC matrix format. Rows correspond to genes in the order provided in features.tsv and columns correspond to cell-barcodes in the order provided in barcodes.tsv.

organism:
Homo sapiens
Mus musculus

source_name: BM, PB, Lung

extract_protocol:
Human CD33+ myeloid cells  were sorted from different tissues of humanized mice, and then labeled with cell hashing antibodies. Cells were loaded into one lane of a 10X Chromium microfluidic chip. Cells capture, lysis and RNA extraction were performed using the 10X V3 Chromium technology following the manufacturer’s protocol.
Single cell capture, barcoding and library preparation were performed with the 10x Chromium system using version 3 chemistry, following the manufacturer’s protocol. cDNA was synthesized and amplified as per manufacturer’s protocol and amplified to construct an Illumina sequencing library. cDNA and libraries were checked for quality on an Agilent 4200 TapeStation, quantified by KAPA qPCR.

value: BM, PB, Lung

tag: cell type

value: Human CD33+ myeloid cells

value: Homozygous

tag: strain


title: Liver_H2O_Inulin13C_RNA_rep3

value: Healthy control mice with 13C-labeled inulin


title: Liver_DSS_Inulin13C_RNA_rep1


title: Monkey_Gallbladder_Female_1501

description:
Monkey_Gallbladder_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: CMTR2 KO CapQuant-seq 2

value: CMTR2 knockout


title: CMTR2 KO CapQuant-seq 1


title: Liver_H2O_Inulin13C_RNA_rep2


title: HEK293T (+) decap CapQuant-seq 1


title: Liver_DSS_InulinUn_RNA_rep3

value: DSS colitis mice with unlabeled inulin


title: Monkey_Gallbladder_Female_1502

description:
Monkey_Gallbladder_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Liver_H2O_Inulin13C_RNA_rep1


title: HEK293T (+) decap CapQuant-seq 2


title: Monkey_FrontalLobe_Female_1501

description:
Monkey_FrontalLobe_Female_1501
Cyno_FPKM_table_Mar22.xlsx

source_name: Frontal Lobe

value: Frontal Lobe


title: HEK293T (-) decap CapQuant-seq 2


title: Monkey_FrontalLobe_Female_1502

description:
Monkey_FrontalLobe_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Monkey_GastrocnemiusTendon_Female_1502

description:
Monkey_GastrocnemiusTendon_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: HEK293T (-) decap CapQuant-seq 1


title: Monkey_FemoralVein_Female_1503

description:
Monkey_FemoralVein_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Femoral Vein

value: Femoral Vein


title: Liver_H2O_InulinUn_RNA_rep3

value: Healthy control mice with unlabeled inulin


title: Fruitfly CapQuant-seq 2

organism: Drosophila melanogaster

source_name: adult fly

growth_protocol: Poly(A)+ RNA extracted from adult flies was purchased from Takara, 636222.

value: whole


title: Monkey_FemoralVein_Female_1502

description:
Monkey_FemoralVein_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Fruitfly CapQuant-seq 1


title: Monkey_FemoralVein_Female_1501

description:
Monkey_FemoralVein_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Colon_DSS_Inulin13C_RNA_rep3

description: processed data file: results_deseq2_colon.csv

source_name: Frozen colon tissue

value: DSS colitis mice with 13C-labeled inulin

value: Frozen colon tissue


title: Monkey_Esophagus_Female_1503

description:
Monkey_Esophagus_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Esophagus

value: Esophagus

value: Female

value: untreated


title: A3-68-LS △rpoS Rep3

city: Hamilton

first: Patricia

last: Rosa

state: MT

address: 903 S 4th St

department: Laboratory of Bacteriology

institute: Rocky Mountain Labs, NIAID, NIH

data_processing:
RNA-seq reads were compiled and filtered to remove any reads with PHRED scores less than 10 and aligned to the B. burgdorferi B31 genome (RefSeq AE000783-AE000794 and AE001575-AE001584) using bowtie2
Reads for annotated genes were determined using featureCounts
Assembly: RefSeq AE000783-AE000794 and AE001575-AE001584
Supplementary files format and content: featureCounts

organism: Borreliella burgdorferi B31

source_name: Bacterial culture

growth_protocol: B. burgdorferi strains were cultured in Barbour-Stoener-Kelly (BSK II) medium supplement with 6% rabbit serum and appropriate antibiotics (streptomycin, 50 μg/mL; kanamycin, 200 μg/mL) at 35⁰C under 2.5% CO2

extract_protocol:
RNA was isolated using TRIzol reagent per the manufacturer’s instructions and treated with 1 unit of DNAse I for 1 hr at 37 C
cDNA libraries were synthesized using Ribo-Zero and ScriptSeq complete bacteria kit with indexing primers for synthesis of directional libraries per the manufacturer’s instructions. The quality and quantity of the resulting cDNA libraries were assessed with an Agilent DNA 1000 assay on an Agilent 2100 Bioanalyzer and with a KAPA Library Quantification Kit prior to RNA-seq.

treatment_protocol: IPTG was either added at a final concentration of 1 mM or ommited from the growth medium

value: Bacterial culture

value: A3-68-LS {delta}rpoS


title: Colon_DSS_Inulin13C_RNA_rep2


title: Monkey_FrontalLobe_Female_1503

description:
Monkey_FrontalLobe_Female_1503
Cyno_FPKM_table_Mar22.xlsx


title: Liver_DSS_InulinUn_RNA_rep2

value: Frozen liver tissue


title: A3-68-LS △rpoS Rep2


title: Liver_DSS_InulinUn_RNA_rep1


title: Monkey_Esophagus_Female_1502

description:
Monkey_Esophagus_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Colon_DSS_Inulin13C_RNA_rep1


title: Monkey_Heart_Female_1503

description:
Monkey_Heart_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Heart

value: Heart


title: Monkey_Duodenum_Female_1503

description:
Monkey_Duodenum_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Duodenum

value: Duodenum


title: Monkey_Esophagus_Female_1501

description:
Monkey_Esophagus_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Colon_H2O_Inulin13C_RNA_rep3

value: Healthy control mice with 13C-labeled inulin


title: flacp::ibbd18△rpoS w/o IPTG Rep3

value: w/o IPTG


title: A3-68-LS △rpoS Rep1


title: Monkey_Duodenum_Female_1502

description:
Monkey_Duodenum_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: flacp::ibbd18△rpoS w/o IPTG Rep2


title: Liver_H2O_InulinUn_RNA_rep2

description: processed data file: results_deseq2_liver.csv


title: flacp::ibbd18△rpoS w/o IPTG Rep1


title: Monkey_Duodenum_Female_1501

description:
Monkey_Duodenum_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Colon_H2O_Inulin13C_RNA_rep2


title: Colon_H2O_Inulin13C_RNA_rep1


title: flacp::ibbd18△rpoS + 1 mM IPTG Rep3

value: 1 mM IPTG


title: Colon_DSS_InulinUn_RNA_rep3

value: DSS colitis mice with unlabeled inulin


title: Colon_DSS_InulinUn_RNA_rep2


title: Liver_H2O_InulinUn_RNA_rep1


title: Monkey_DorsalRootGanglia_Female_1503

description:
Monkey_DorsalRootGanglia_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Ganglia, Spinal

value: Ganglia, Spinal


title: flacp::ibbd18△rpoS + 1 mM IPTG Rep2


title: Monkey_DorsalRootGanglia_Female_1502

description:
Monkey_DorsalRootGanglia_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: flacp::ibbd18△rpoS + 1 mM IPTG Rep1


title: Monkey_DorsalRootGanglia_Female_1501

description:
Monkey_DorsalRootGanglia_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: A3-68-LS w/o IPTG Rep3


title: Monkey_Cornea_Female_1503

description:
Monkey_Cornea_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Cornea

value: Cornea


title: Colon_H2O_InulinUn_RNA_rep2

value: Healthy control mice with unlabeled inulin


title: Monkey_Cornea_Female_1502

description:
Monkey_Cornea_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Colon_H2O_InulinUn_RNA_rep1


title: A3-68-LS w/o IPTG Rep2


title: A3-68-LS w/o IPTG Rep1


title: Monkey_Colon_Female_1503

description:
Monkey_Colon_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Colon

value: Colon


title: Cecum_DSS_Inulin13C_RNA_rep3

description: processed data file: results_deseq2_cecum.csv

source_name: Frozen cecal tissue

value: DSS colitis mice with 13C-labeled inulin

value: Frozen cecal tissue


title: Cecum_DSS_Inulin13C_RNA_rep2


title: Monkey_Colon_Female_1502

description:
Monkey_Colon_Female_1502
Cyno_FPKM_table_Mar22.xlsx

value: Female


title: Monkey_Colon_Female_1501

description:
Monkey_Colon_Female_1501
Cyno_FPKM_table_Mar22.xlsx

value: untreated


title: A3-68-LS + 1 mM IPTG Rep1

value: Bacterial culture


title: Cecum_DSS_Inulin13C_RNA_rep1


title: Cecum_H2O_Inulin13C_RNA_rep3


title: Cecum_H2O_Inulin13C_RNA_rep2


title: Monkey_Cervix_Female_1502

description:
Monkey_Cervix_Female_1502
Cyno_FPKM_table_Mar22.xlsx

source_name: Cervix Uteri

value: Cervix Uteri


title: Colon_DSS_InulinUn_RNA_rep1

description: processed data file: results_deseq2_colon.csv

value: Frozen colon tissue


title: flacp::ibbd18(rec) Rep2


title: flacp::ibbd18(rec) Rep1


title: Colon_H2O_InulinUn_RNA_rep3


title: Cecum_H2O_Inulin13C_RNA_rep1


title: Cecum_DSS_InulinUn_RNA_rep3


title: flacp::ibbd18 w/o IPTG Rep3


title: Monkey_CervicalSpinalCord_Female_1503

description:
Monkey_CervicalSpinalCord_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Cervical Cord

value: Cervical Cord


title: Monkey_Cornea_Female_1501

description:
Monkey_Cornea_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Cecum_DSS_InulinUn_RNA_rep2


title: Monkey_Cervix_Female_1501

description:
Monkey_Cervix_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Monkey_CervicalSpinalCord_Female_1502

description:
Monkey_CervicalSpinalCord_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: A3-68-LS + 1 mM IPTG Rep3


title: A3-68-LS + 1 mM IPTG Rep2


title: Monkey_CervicalSpinalCord_Female_1501

description:
Monkey_CervicalSpinalCord_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Cecum_DSS_InulinUn_RNA_rep1


title: flacp::ibbd18(rec) Rep3


title: flacp::ibbd18 w/o IPTG Rep1


title: flacp::ibbd18 + 1 mM IPTG Rep3


title: Monkey_Cervix_Female_1503

description:
Monkey_Cervix_Female_1503
Cyno_FPKM_table_Mar22.xlsx


title: Monkey_Cerebellum_Female_1503

description:
Monkey_Cerebellum_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Cerebellum

value: Cerebellum


title: Monkey_Cerebellum_Female_1502

description:
Monkey_Cerebellum_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: flacp::ibbd18 + 1 mM IPTG Rep2


title: Cecum_H2O_InulinUn_RNA_rep2


title: Cecum_H2O_InulinUn_RNA_rep1


title: Monkey_Cerebellum_Female_1501

description:
Monkey_Cerebellum_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Adh excess C

city: Charlottesville

first: Eugene

last: Ke

state: Virginia

address: P.O. Box 800734, 1340 Jefferson Park Avenue

department: MIC

institute: University of Virginia

data_processing:
Reads were checked for quality using FASTQC (v0.11.8)
Trimmed using BBMAP (v3.8.16b)
Aligned to the Neisseria gonorrhoeae FA1090 genome using STAR (v2.7.1a)
Transcripts per million calculations were performed by RSEM (v1.3.1)
Genome_build: FA1090 genome with Ensembl annotations (ASM684v1.51)
Supplementary_files_format_and_content: tab delimited output files containing gene summarized transcripts per million as outputted by RSEM

organism: Neisseria gonorrhoeae

growth_protocol: Suspension-grown: N. gonorrhoeae (FA1090 1-81-S2 OpaD+nv (Ball and Criss J Bacteriol 2013)) was prepared in experimental media described in "treatment protocol", inoculated at 1x10^8 CFU/mL into 96-well round-bottom plates (Sarstedt 82.1582.001), and grown for 4 h at 37 °C in 5% CO2. Adherent: N. gonorrhoeae (10^8 CFU/ml in 500 μL KSFM) were grown and allowed to adhere to 18 mm diameter acid-washed glass coverslips (Thermo Fisher #12-545-100P) in 12-well plates for 2 h, then washed to remove non-adherent bacteria. The experimental media (described in treatment protocol) was added and the infection was allowed to proceed for another 4 h at 37 °C in 5% CO2.

extract_protocol:
Suspension: Bacteria from 16 wells were pooled together in a 15mL conical tube, and two volumes of RNAprotect bacteria reagent (Qiagen 76506) were added. Adherent: supernatant was removed and 1 volume (500 μL) KSFM-GF and 2 volumes (1 mL) of RNAprotect bacteria reagent (Qiagen 76506) were added. N. gonorrhoeae was collected by scraping, and bacteria from 8 wells were pooled together in a 15mL conical tube. Both: bacteria were incubated at room temperature for 5 min, and pelleted at 4,000 x g for 11 min.Samples were lysed according to the “Enzymatic Lysis and Proteinase K Digestion of Bacteria” protocol from Qiagen before proceeding with RNeasy Plus mini kit (Qiagen 74134) per manufacturer’s instructions.
Ribosomal RNA reduction was completed using NEBNext rRNA Depletion Kit (Bacteria) (New England Biolabs E7850), and RNA library prep was done using NEBNext Ultra II Directional RNA Library Prep Kit (NEB E7760).

treatment_protocol: Keratinocyte serum free medium (KSFM) was obtained from Gibco (17005042) and prepared without the addition of BSA and EGF. KSFM was incubated for 20 min at 37 °C with no additives (normal), with 3 μM recombinant murine calprotectin (mCp) to sequester free zinc (sequestered), or with 3 μM murine calprotectin and 6 μM ZnSO4 (excess).

tag: substrate

value: Adherent

value: KSFM with 3 μM mCp and 6 μM ZnSO4


title: Monkey_CaudateNucleus_Female_1503

description:
Monkey_CaudateNucleus_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Caudate Nucleus

value: Caudate Nucleus


title: Conv_input_rep1

library_source: genomic

molecule: genomic DNA

source_name: Colonic epithelial cells

extract_protocol:
To prepare epithelial cell pellets, colons were incubated in PBS containing 30 mM EDTA and 1.5 mM DTT for 20 minutes at 4°C with occasional inversion.  Tissue was subsequently transferred to PBS containing 30 mM EDTA, incubated for 10 mins at 37°C, and shaken vigorously for 1-2 minutes until the mesenchyme separated from the epithelium.  The mesenchyme was removed and epithelial cells were pelleted at 1500 rpm for 5 minutes at 4°C and washed in PBS.  Cell pellets were further incubated with dispase (Corning, 0.3 U/ml) in PBS (100 l dispase per 50 ml PBS) at 37°C for 10 mins and then quenched with 1/10 volume of FBS containing DNase followed by washing in PBS.  Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until use.  For ChIP-seq, cells were cross-linked prior to freezing by treatment with 1% formaldehyde in PBS for 5 mins at room temperature, followed by quenching with 125 mM glycine for an additional 5 mins at room temperature.  Cross-linked pellets were then washed in PBS and flash frozen until use.    Nuclear lysates were prepared by resuspending cells in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x protease inhibitors) and rotating for 10 mins at 4°C.  After centrifugation at 3000 rpm for 5 mins at 4°C, the cell pellet was resuspended in lysis buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x protease inhibitors) and rotated for 10 mins at room temperature.  Cells were pelleted and then resuspended in lysis buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mm EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.1% N-lauroylsarcosine, 1x protease inhibitors) for chromatin shearing with a Covaris S220 focused ultrasonicator (8.4W, 200 peak power, 200 cycles/burst, 900 seconds) in a 4°C water bath.  After sonication, Triton X-100 was added to 1% final concentration and the lysate was cleared by centrifugation at top speed in a microfuge for 10 mins at 4°C.  Magnetic protein G beads were washed with blocking solution (0.5% BSA in PBS) and then incubated with rabbit anti-serum against acetylated histone H4 (Millipore 06-866) in blocking solution for several hours at 4°C with rotation.  Beads were then washed in blocking solution and then added to nuclear lysates containing a spike-in of Drosophila S2 chromatin (cells generously provided by P. Pascual-Garcia), prepared as above.   After overnight incubation at 4°C, beads with captured chromatin were washed with 3 x 1 ml RIPA wash buffer (50 mM HEPES-KOH pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate) and 1 x 1 ml of final wash buffer (10 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA).  Beads were eluted with 200 µl elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) for 30 mins at 65°C.  The supernatant was then incubated at 65°C overnight to reverse cross-links.  After dilution with one volume of TE, RNase was added to 0.2 mg/ml and incubated at 37°C for 2 hrs.  Proteinase K was then added to 0.2 mg/ml and incubated at 55°C for 2 hrs.  DNA was purified by phenol-chloroform extraction with subsequent ethanol precipitation.
Purified DNA was resuspended in 10 mM Tris pH 8 and prepared for sequencing using the NEBNext Ultra II DNA library kit for Illumina (New England Biolabs).

treatment_protocol: Conventional mice (C57BL/6J, female, 8 weeks) were purchased from Jackson Laboratories.  Germ-free mice (C57BL/6J, female, 7-8 weeks) were provided by the Penn Gnotobiotic Mouse Facility.  Animals were euthanized by CO2 asphyxiation followed by cervical dislocation.  Colons and ceca were dissected, flushed with sterile phosphate-buffered saline (PBS), splayed open longitudinally, and washed again with PBS for use in epithelial cell isolation.

value: Conventional mice

value: Colonic epithelial cells


title: Adh excess B


title: Conv_input_rep2


title: Monkey_CaudateNucleus_Female_1502

description:
Monkey_CaudateNucleus_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: flacp::ibbd18 w/o IPTG Rep2

value: w/o IPTG


title: Monkey_CaudateNucleus_Female_1501

description:
Monkey_CaudateNucleus_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Cecum_H2O_InulinUn_RNA_rep3

description: processed data file: results_deseq2_cecum.csv

value: Healthy control mice with unlabeled inulin


title: Adh sequestered C

value: KSFM with 3 μM mCp


title: Adh excess A


title: Conv_H4ac4_rep1

tag: chip antibody

value: Anti-acetyl histone H4 (H4ac4, Millipore 06-866)


title: Monkey_BuccalMucosa_Female_1503

description:
Monkey_BuccalMucosa_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Mouth Mucosa

value: Mouth Mucosa


title: GF_input_rep2

value: Germ-free mice


title: Monkey_BuccalMucosa_Female_1502

description:
Monkey_BuccalMucosa_Female_1502
Cyno_FPKM_table_Mar22.xlsx

value: Female

value: untreated


title: Adh sequestered B

value: Bacterial culture


title: Monkey_BuccalMucosa_Female_1501

description:
Monkey_BuccalMucosa_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Adh normal C

value: KSFM


title: Adh sequestered A


title: GF_input_rep1


title: GF_H4ac4_rep1


title: Monkey_BoneMarrow_Female_1502

description:
Monkey_BoneMarrow_Female_1502
Cyno_FPKM_table_Mar22.xlsx

source_name: Bone Marrow

value: Bone Marrow


title: GF_H4ac4_rep2


title: flacp::ibbd18 + 1 mM IPTG Rep1

value: 1 mM IPTG


title: Adh normal A

value: Adherent


title: Conv_RNA_rep5

description: processed data file: gf_results_deseq2.csv

extract_protocol:
To prepare epithelial cell pellets, colons were incubated in PBS containing 30 mM EDTA and 1.5 mM DTT for 20 minutes at 4°C with occasional inversion.  Tissue was subsequently transferred to PBS containing 30 mM EDTA, incubated for 10 mins at 37°C, and shaken vigorously for 1-2 minutes until the mesenchyme separated from the epithelium.  The mesenchyme was removed and epithelial cells were pelleted at 1500 rpm for 5 minutes at 4°C and washed in PBS.  Cell pellets were further incubated with dispase (Corning, 0.3 U/ml) in PBS (100 l dispase per 50 ml PBS) at 37°C for 10 mins and then quenched with 1/10 volume of FBS containing DNase followed by washing in PBS.  Cell pellets were flash frozen in liquid nitrogen and stored at -80°C until use.    Total RNA was extracted from epithelial cell pellets using the RNeasy kit (Qiagen) with QIAshredder columns.
The NEBNext Ultra or Ultra II Directional RNA Library Prep Kit for Illumina with the mRNA magnetic isolation module (New England Biolabs) was used to prepare sequencing libraries from 1 µg of total RNA.


title: Monkey_BoneMarrow_Female_1503

description:
Monkey_BoneMarrow_Female_1503
Cyno_FPKM_table_Mar22.xlsx


title: Adh normal B


title: Monkey_BoneMarrow_Female_1501

description:
Monkey_BoneMarrow_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Conv_RNA_rep4


title: Conv_H4ac4_rep2


title: Sus excess C

value: Suspension

value: KSFM with 3 μM mCp and 6 μM ZnSO4


title: Monkey_Aorta_Female_1503

description:
Monkey_Aorta_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Aorta

value: Aorta


title: Sus excess B


title: Monkey_Aorta_Female_1502

description:
Monkey_Aorta_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: Conv_RNA_rep3

value: Conventional mice

value: Colonic epithelial cells


title: Conv_RNA_rep2


title: p53-R273H + ATO

city: Shanghai

first: Yigang

last: Tang

address: No 197 RUIJIN ER RD

country: China

institute: Shanghai Jiaotong University

data_processing:
Raw sequence reads were initially processed using FastQC (Babraham Institute, Cambridge, UK) for quality control, then adapter sequences and poor-quality reads were removed using Cutadapt.
Quality-filtered reads were then mapped to the human genome using STAR software, and only uniquely mapped reads were kept.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files includes FPKM value of each sample

growth_protocol: HCT116 p53-/- cells were cultured at 37 ℃ in medium suggested in the repository, supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin.

extract_protocol:
Total RNA was isolated using a Total RNA Purification Kit (B518651, Sangon Biotech)
Sequencing libraries was constructed from 1 μg of purified mRNA using an Illumina TruSeq RNA Sample Prep Kit (cat# FC-122-1001)
Libraries were pooled and 150-bp paired-end reads were sequenced on the Illumina HiSeq Xten platform.

treatment_protocol: HCT116 p53-/- cells infected with vehicle, p53-R273H, or p53-M133T were treated with 1 μg ml-1 ATO for 24 h.

tag: cell line background

tag: genotype/variation

value: HCT116 p53-/- cells  infected with p53-R273H

tag: source tissue

value: colon

value: 1 μg ml-1 ATO for 24 h


title: Monkey_Aorta_Female_1501

description:
Monkey_Aorta_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Sus sequestered C

value: KSFM with 3 μM mCp


title: p53-R273H


title: Sus excess A


title: Conv_RNA_rep1


title: GF_RNA_rep5

value: Germ-free mice


title: p53-M133T + ATO

value: HCT116 p53-/- cells  infected with p53-M133T


title: Monkey_AdrenalGland_Female_1502

description:
Monkey_AdrenalGland_Female_1502
Cyno_FPKM_table_Mar22.xlsx

source_name: Adrenal Glands

value: Adrenal Glands

value: Female

value: untreated


title: p53-M133T


title: Monkey_AdrenalGland_Female_1501

description:
Monkey_AdrenalGland_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Vechile + ATO

source_name: HCT116 p53-/-_Vechile + ATO

value: HCT116 p53-/- cells


title: Sus sequestered B

source_name: Bacterial culture

value: Bacterial culture


title: Sus sequestered A


title: GF_RNA_rep3


title: Monkey_AdiposeFat_Female_1503

description:
Monkey_AdiposeFat_Female_1503
Cyno_FPKM_table_Mar22.xlsx

source_name: Adipose Tissue

value: Adipose Tissue


title: Sus normal C

value: Suspension

value: KSFM


title: GF_RNA_rep2


title: Sus normal B


title: Vechile

source_name: HCT116 p53-/-_Vechile


title: Monkey_AdiposeFat_Female_1502

description:
Monkey_AdiposeFat_Female_1502
Cyno_FPKM_table_Mar22.xlsx


title: GF_RNA_rep1


title: Wild type p53

source_name: HCT116 p53-/-_Wild type p53

value: HCT116 p53-/- cells  infected with wild type p53


title: Sus normal A


title: Monkey_AdrenalGland_Female_1503

description:
Monkey_AdrenalGland_Female_1503
Cyno_FPKM_table_Mar22.xlsx


title: Monkey_AdiposeFat_Female_1501

description:
Monkey_AdiposeFat_Female_1501
Cyno_FPKM_table_Mar22.xlsx


title: Xyl_SS

status: Public on Dec 11 2022

city: Tianjin

first: Feiyu

last: Fan

state: Tianjin

address: 32 West 7th Avenue, Tianjin Airport Economic Area

institute: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Cyberlindnera jadinii NBRC0988 genome using HISAT 2 (v2.2.1)
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated
Detailed parameters for reads mapping: hisat2 -p 8 -x CJ --rna-strandness RF --max-intronlen 1000 --no-mixed --no-discordant -1 SSGlu_1.clean.fq.gz -2 SSGlu_2.clean.fq.gz -S SSGlu.sam
Assembly: Cyberlindnera jadinii NBRC0988 (Accession numbers： BAEL01000001-BAEL01001163 and DG000065-DG000077)
Supplementary files format and content: Tab-delimited text files include FPKM values for each Sample .

organism: Cyberlindnera jadinii NBRC 0988

source_name: Yeast cells

growth_protocol: NBRC0988 cells grown overnight were inoculated into 100 ml liquid Yeast Extract Peptone Dextrose (YEPD) medium with the initial inoculation amount of OD600= 0.1, and cultured for 15 hours to the logarithmic growth phase (OD600= 2-3).

extract_protocol:
Total RNA was isolated by the Trizol-Phenol-Chloroform method. An additional clean-up including the on-column DNase I treatment was performed by using the RNeasy mini kit (Qiagen).
RNA libraries were prepared for sequencing using standard Illumina protocols. 150-nt paired-end strand-specific RNA-seq libraries (SS_lib_type RF)

treatment_protocol: The cells were collected by centrifugation at 6,000g for 5 minutes. After washing twice with phosphate buffer saline (PBS), they were inoculated into new 100 mL YEP medium containing 2% w/v glucose or xylose.After flask culturing at 30°C and 250 rpm for an additional 5 hours, the yeast cells were collected by centrifugation for total RNA isolation and Illumina RNA-seq library construction.

value: Yeast cells

value: WT

value: xylose


title: Glu_SS

value: glucose


title: GF_RNA_rep4

value: Colonic epithelial cells


title: sc_rep_mEB_series_oBC_CS1_repA_lane2_reseq

city: Montreal

first: Jean-Benoit

last: Lalanne

state: QC

address: 5155 Chem. de la Rampe

department: Biochemistry and Molecular Medicine

country: Canada

institute: University of Montreal

description:
10X Genomics (custom oBC library)
oBC_counts_sc_rep_mEB_series.txt
assigned_oBC_CRE_mBC_joined_counts_sc_rep_mEB_series.txt

library_source: transcriptomic single cell

data_processing:
GEx libraries: Fastq files were generated using the makefastq command from cellranger (v6.0.1), and the gene expression count matrices were then generated with cellranger count command, with transcriptome reference mm10-3.0.0. Raw count matrices were then imported as a Seurat object (filtering gene expressed in less than 3 cells, and cell barcodes with less than 50 genes measured). Cell barcodes in the high total UMI mode with low mitochondrial RNA proportion were filtered as likely bona fide cells (fraction of mitochondrial UMI >1% and <15%, total gene expression UMI > 400 for samples from replicates A, B, and 2B lane1, and >1000 for 2B lane2, which was fortuitously sequenced more deeply).  The filtered count matrices were then used to evaluate doublet scores using scrublet (scrub_doublets command, 30 principal components, mean_center=true, normalize_variance=true), and cell barcodes with doublet score > 0.3 (separating the two modes of the simulated doublet distribution from scrublet) were filtered out. Datasets from all replicates were then combined in a single Seurat object, dimensionally reduced and clustered (NormalizeData, normalization.method= “LogNormalize”, scale.factor=10000; FindVariableFeatures with selection.method = “vst”, nfeatures=1000; ScaleData with all genes as features; RunPCA with identified variable features and 100 principal components; FindNeighbors, dims=1:50; FindClusters, resolution=0.2; RunUMAP, dims=1:50, n.neighbors=50) without batch correction given the good correspondence between replicates (Fig. S8B). The cluster identities were taken as categories for cell-type expression testing (see integration section below).
The following additional quality filtering steps were applied to retain high confidence singlet cells. Clusters comprising less than 1% of cells were considered likely doublets/artifacts, and corresponding cells were removed. Cells members of each cluster identified were separately sub-clustered with the same procedure as above (except resolution 0.5 in FindNeighbors). Any sub-cluster with a median doublet score above 0.15 was deemed composed of likely doublets, and corresponding cells were removed. Cells with anomalously high gene expression UMI counts were removed (with 10x lane specific thresholds: >10k for A.1, >9k A.2, >12k B.1, 9k B.2, <15k 2B.2, no such cells in 2B.1). Finally, cells with an estimated MOI > 200 (roughly corresponding to the top 0.1% of the distribution, MOI estimated through oBC UMI > 10, see below) were filtered out. In the end, n=43799 cells passed all these quality filters (12859 replicate A, 15422 replicate B, 15518 replicate 2B).
mBC libraries: Data was converted to fastq using bcl2fastq, and fastqs were minimally processed (e.g., trimming read 1 to 28 cycles) to be compatible with cellranger (version 6.0.1, 10x Genomics), which was run to perform error correction on cell barcodes. The resulting position sorted bam files were then parsed for the mBC reads as follows using a custom python script. Reads aligning to the reference genome or without either corrected cell barcode or UMI (tags CB and UB in the bam file) were discarded. Only reads with the exact expected 7 nt sequence (TCGACAA) downstream of the mBC (positions 16 to 22) were retained. List of all UMIs corresponding to a cell barcode and mBC pair were stored, discarding chimeric UMIs (taken to be UMIs for which the proportion of reads associated to a given mBC vs all other mBC in the specified cell barcode falls below 0.2). mBC comprised of all Gs (empty read) were discarded. Finally, the UMI count was error corrected as follows. For each given mBC and cell barcode, the Hamming distance between all UMIs was calculated, a graph created by connecting UMIs that were a Hamming distance ≤ 1, and the resulting the number of connected components in the graph was taken as the error-corrected UMI count for a given cell barcode-mBC pair. These error corrected UMI counts were used for the per single-cell quantification of the reporter mRNA expression.
oBC libraries: processed in an entirely analogous way to the strategy for mBC, with the following modifications: two sequencing runs were combined in a single fastq prior to processing, read 2 were trimmed to 23 cycles, and only reads with the GCTTTAA (constant region after the oBC) at positions 17 to 23 were retained. The number of UMIs per oBC per cell barcode was also taken as the error corrected (1 Hamming distance) count and our measure of oBC expression in single cells (see below for a normalization strategy to correct for gene expression UMIs).  Given that cell barcodes derived from capture sequence vs. poly-dT reverse transcription primer are different (bases 8 and 9 reverse complemented) on the same bead (and not error corrected by cellranger in our application), we converted the CS1 cell barcode to its poly-dT counterpart to enable matching across the different libraries.
Only cell barcodes passing the QC filters from the GEx analysis were retained in the final count tables. Only mBC and oBC present in the subassembly library were retained.
Assembly: mm10
Supplementary files format and content: The four processed files are:
Supplementary files format and content: Seurat processed object containing transcriptome quantification and cell annotation metadata (combined for all replicates).
Supplementary files format and content: Raw oBC count table (per cellbarcode, restricted to oBC from the list determined in the subassembly and UMI counts > 1).  Column 1: cell barcode; column 2: replicate ID; column 3: oBC; column 4: read counts
Supplementary files format and content: Raw mBC count table (per cellbarcode, restricted to mBC from the list determined in the subassembly). Column 1: cell barcode; column 2: replicate ID; column 3: mBC; column 4: read counts; column 5: UMI counts; capture modality.
Supplementary files format and content: Final assigned joined cell-oBC-CRE_mBC table. Restricting to oBC with >10 UMI counts, and to uniquely matchable oBC-CRE-mBC triplets. column 1: cell barcode; column 2:  replicate ID; column 3: oBC; column 4: mBC; column 5: CRE class (promoters [exogenous series]  or devCRE); column 6: CRE identity; column 7: read counts oBC; column 8: UMI counts oBC; column 9: read counts mBC; column 10: UMI counts mBC

source_name: WD44 (mouse embryonic stem cells)

growth_protocol:
Following integration of reporters via piggyBac (cells growing for >14 days for plasmid dilution), exponentially growing mESCs are lifted from the plate (aspirate medium, wash with PBS, add 2.5 mL [for 10 cm plate] 0.05% trypsin , incubate 2 minutes at 37C, deactivate trypsin and triturate to a single-cell suspension with 10 mL pre-warmed SL medium). Cells are then counted and spun down (5 min at 300 g). Supernatant is aspirated and cells are resuspended to 2 M/mL in CA medium (medium for EB induction: DMEM, 10% FBS, 1x MEM non-essential amino-acids, 1x Glutamax , 10-5 beta-mercaptoethanol). Cells are counted again, and density adjusted to 1 M/mL with CA medium. 3 mL (3 M cells) are added to 12 mL of CA medium in 10 cm plates (non gelatinized, non adherent). One the next day, plates are gently agitated to promote cell aggregation.
Following induction, embryoid bodies (mEBs) are passaged every two days (no daily medium change). mEBs are collected using a serological pipette and transferred to a 50 mL conical tube (typically three plates are pooled). Leftover mEBs on plates are recovered by a CA medium wash and pooled with in the conical tube. mEBs are left to settle (initially up to 15-20 min, faster as the mEBs grow in size). Once mEBs have settled, medium is aspirated from the top, carefully avoiding disturbing the loose pellet. Fresh, pre-warmed, CA medium is then added to 15 mL/plate and mEBs redistributed to plates.

extract_protocol:
mEBs were processed at the three weeks end point as follows (for each replicate): 2 plates of mEBs were pooled into a 50 mL conical left to settle. Medium was aspirated and mEBs were washed twice with PBS, resuspended in 3 mL PBS in the second wash, and split in two 1.5 mL aliquots in 2 mL tubes. PBS was aspirated from the tubes, and 500 uL of trypsin 0.25% was added per tube. Tubes were then mixed on a thermomixer at 37C and 650 rpm for 4 minutes. Cells were then gently dissociated by pipetting up and down 10 times, and placed back on the thermomixer for 2 min. 1 mL of SL medium was then added per sample and pipetted to obtain a single-cell suspensions, the two samples were combined in a 15 mL conical, and passed through a 100 um strainer. The strained single-cell suspension was counted, and cells were spun down (300 g, 5 min), resuspended to 4 M/mL, and taken to FACS. >600k  cells were then FACS sorted (in <50 min) in pre-warmed SL medium to ensure the single-cell nature of the suspension (no gating on fluorescence proteins) prior to generating the emulsions for single-cell RNA-seq. Sorted cells were then spun down at 400 g at 4C for 5 min, the medium gently aspirated, and resuspended to an expected 2.5 M cells/mL (based on FACS sort event counts) in ice cold PBS + 0.04% BSA, cells were counted and volume adjusted to 1200 k/uL with ice cold PBS+BSA.
Single-cell suspensions in PBS+BSA were taken as the starting point for the 10x Genomics protocol (v3.1 with feature barcoding). Emulsion and reverse transcription were performed per the manufacturer’s instruction. Given prior empirical experience with mEBs processing, each 10x lane was slightly overloaded (by an additional 20%) to approach the expected recovery of 10k cells/lane. Each replicate was profiled with 2 lanes of 10x, for a total of 6 lanes.
For single-cell reporters, three libraries are generated: the standard 3’ gene expression library from 10x (GEx), and two custom derived libraries, one for each reporter RNA (oBC and mBC), obtained from nested PCRs from the amplified cDNA. Briefly, Single-cell library preparation proceeded following the manufacturer's protocol (v3.1 manual CG000205 Rev D, 10x Genomics), with some modifications. For cDNA amplification, primers specific to the mBC (oSR38) and oBC (o246) reporter transcripts were spiked-in the reaction (similar to TAP-seq) at final concentration of 0.5 uM to boost capture. Following cDNA amplification, both the bead and supernatant derived material (steps 2.3Ax and 2.3Bxiv respectively) were saved for downstream processing.
Gene expression libraries for all replicates were prepared following the manufacturer’s protocol from 25% of the bead fraction amplified cDNA.
oBC enriched libraries were prepared as follows. 25% of the supernatant fraction from the amplified cDNA was taken as input for semi-nested inner PCR was performed with Kapa Robust (50 uL 2x master mix, supernatant cDNA, 5 μL 10 μM NextP5_index1 primer, 5 μL 10 μM indexed primers [o501-o506, one per sample], 0.5 uL SYBr green, and water to 100 μL; run parameters: 3 min at 95C, and cycles 20 s at 95C, 20 s at 60C, 20 s at 72C) with tracking with qPCR and stopped before the inflection point. Libraries were purified by 1.5x ampure. To avoid loop-the-loop products in the oBC libraries, the lowest band in the circularized ladder amplicons was size selected on PAGE (6% TBE, 180V, 30 min) for each library and used for sequencing.
Only the poly-dT captured libraries were generated for the mBC for the mouse embryoid body experiment. 25% of the bead-fraction of the purified amplified cDNA was used as template for PCR with Kapa Robust (50 uL 2x master mix, supernatant cDNA, 5 μL 10 μM o324 primer, 5 μL 10 μM o529, 0.5 uL SYBr green, and water to 100 μL; run parameters: 3 min at 95C, and cycles 20 s at 95C, 20 s at 65C, 50 s at 72C), with tracking by qPCR and purifying by 1x ampure. A final PCR (same condition as above) was performed to index amplicons with primers o076 and P7-indexed primers (o530-o533), and the resulting amplicons purified by 1x ampure.
Read structures:
RepA/B GEx: read 1, cell-barcode+UMI (28 cycles); index 1, library index (10 cycles); read 2, transcriptome (54 cycles)
RepA/B oBC: read 1, cell-barcode+UMI (28 cycles, no custom primers); index 1, library index (10 cycles, primer o432); read 2, oBC (54 cycles, primers o433)
RepA/B oBC (reseq): read 1, cell-barcode+UMI (28 cycles, primer SR40); index 1, library index (10 cycles, primer o432); read 2, oBC (20 cycles, primers o433)
RepA/B mBC: read 1, cell-barcode+UMI (28 cycles, no custom primers); index 1, library index(10 cycles, primer o534); read 2: mBC (54 cycles, o334)
Rep2B GEx: read 1, cell-barcode+UMI (28 cycles); index 1, library index (8 cycles); read 2, transcriptome (56 cycles)
Rep2B oBC: read 1, cell-barcode+UMI (28 cycles, no custom primers); index 1, library index (8 cycles, primer o432); read 2, oBC (56 cycles, primer o433)
Rep2B mBC: read 1, cell-barcode+UMI (28 cycles, no custom primers); index 1, library index (8 cycles, primer o534); read 2: mBC (56 cycles, primer o334)
scRNA-seq (with custom reporter libraries)

value: WD44 (mouse embryonic stem cells)

value: Mouse embryoid bodies

value: monoclonal CRISPRi (dCAS9-KRAB) cell line, high MOI (polyclonal) single-cell reporter cassette (89% devCRE library + 10% promoter series + 1% EEF1A1-mCherry) delivered by piggyBac

tag: time

value: Day 21


title: sc_rep_mEB_series_oBC_CS1_repB_lane1_reseq


title: sc_rep_mEB_series_oBC_CS1_repB_lane2_reseq


title: sc_rep_mEB_series_oBC_CS1_repA_lane1_reseq


title: bulk_MPRA_mEB_series_mBC_repB_d20_RNA

description: Bulk MPRA

data_processing:
Summary count table per mBC for bulk MPRA data (time series, mouse embryoid bodies). Column 1: mBC; column 2: CRE identity; column 3: CRE class ([exogenous] promoters, devCRE); column 4: replicate ID; column 5: time point (day); column 6: preparation batch id; column 7: DNA pseudo UMI counts; column 8: RNA pseudo UMI counts; column 9: DNA read counts; column 10 RNA read counts. mBC with NA the counts were not detected in the respect experiments (=0)
Sample replicate 2B (tech rep 1) from day 20 was not included because it had clear evidence of genomic DNA signal in the RNA libraries (minimal and no promoter activity >100 higher than all other samples) likely as a result of the low RNA yield.
Supplementary files format and content: bulk_MPRA_counts_mEB_series: read and UMI counts for MPRA for each uniquely mappable barcode (including to exogenous promoters and CREs). Column 1: mBC sequence; column 2: CRE identity; column 3: CRE class (exogenous promoters: promoters, devCRE: putative CREs selected from mouse); column 4: replicate identity; column5: time point (e.g., day 6 = d06); column 6: technical library preparation batch identity; column 7: UMI counts DNA; column 8: UMI counts RNA; column 9: read counts DNA; column 10: read counts RNA.

extract_protocol:
Following induction, mEBs were passaged every two days, with sampling 5-10% of EBs at each time point for bulk MPRA (EBs were pelleted at 5 min at 300 g, medium aspirated, fixed with ice cold 80% methanol, and stored at -80C until processing). Genomic DNA and RNA were extracted with the AllPrep kit (Qiagen). 40 samples (different replicate/batch/time points) were processed overall, comprising 13 samples repA and repB across 12 time point (day 0, 4, 6, 10, 12, 14, 16, 18, 20, 21; with two technical replicates for day 16) and 14 samples for rep2B (two technical replicates for day 0, 8, 12, 16, 18, 20; one replicate each for day 4, 6). From each sample, 2 libraries (1 gDNA-derived, 1 RNA derived) were constructed, for a total of 80 libraries. Libraries were prepared in three batches (batch1: replicates A and B, days 0, 4, 8, 12, 16; batch2: replicates A andB, days 2, 6, 10, 14, 16, 18, 20, 21; batch3: all samples from replicate 2B). For RNA, DNase treatment was applied to the first batch, but was found to be unnecessary (comparing with no reverse transcription controls), and was consequently not performed on the other two batches.
Amplicon libraries from DNA were generated in two steps of PCR amplification with Kapa HiFi (Roche). 0.5-1 ug of genomic DNA input was used. For low-cycle number PCR1, gDNA was mixed with 50 μL 2⨉ Kapa HiFi master mix, 5 μL 10 μM o039, 5 μL 10 μM o358, and water to 100 μL. Cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Primer o358 contains 10 random Ns to serve as a pseudo-UMI (hereafter referred to as UMIs for brevity) to correct for PCR jackpotting. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. Illumina adapters and sequencing indices were appended through PCR2, with 4 μL of the eluate from PCR1 taken as input, and 25 μL 2⨉ Kapa HiFi master mix, 0.25 μL 100⨉ SYBr green, 2.5 μL 10 μM o077, 2.5 μL 10 μM indexed primers (one of o359-o366, o437-o448, o555-o564 per sample), and water to 50 μL. Libraries were amplified with tracking by qPCR with: 1 min at 95C, and cycles up to the qPCR inflection point: 20 s at 98C, 20 s at 60C, 30 s at 72C. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Amplicons libraries for RNA: 1 μg of RNA was taken to reverse transcription. 2 μL (500 ng/μL) RNA was mixed with 2 μL 1 μM o358, incubated at 65C for 5 min, and placed on ice. 15 μL of reverse transcription master mix was then added (4 μL 5⨉ FS buffer, 1 μL 0.1 M DTT, 1 μL 10 mM dNTP mix, 8 μL water, 1 μL SSIII [Thermo Fisher]), and the reaction incubated at 55C for 60 min, followed by 70C for 15 min. Half of the reverse transcription reaction was then directly amplified for PCR1 (37.5 2⨉ Kapa HiFi master mix, 3.75 μL o039 10 uM, 3.75 μL o077 10 uM, water to 75 μL), with cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. PCR2 proceeded as for libraries prepared from genomic DNA, with o077 and indexing primers (one of o359-o366, o437-o448, o555-o564 per sample), and reactions were stopped at inflexion point from qPCR tracking. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Each preparation batch was sequenced separately.
Batch 1: Nextseq500; read 1, mBC forward (28 cycles, primer o369); index 1, UMI (8 cycles, primer o435); read 2, mBC reverse (43 cycles, primer o371); index 2, library index (6 cycles, primer o370).
Batch 2: Nextseq2000, same set of primers (well 1: o369+o370, well2: o370+o435). read 1, mBC forward (28 cycles); index 1, UMI (10 cycles); read 2, mBC reverse (20 cycles); index 2, library index (6 cycles).
Batch 3: Nextseq500; read 1, mBC forward (18 cycles, primer o369); index 1, UMI (10 cycles, primer o435); read 2, mBC reverse (20 cycles, primer o371); index 2, library index (6 cycles, primer o370)."
Bulk MPRA

tag: day

value: Day 20


title: bulk_MPRA_mEB_series_mBC_repB_d16_RNA_extract_lib_rep

description: Bulk MPRA

tag: tissue

value: Day 16


title: bulk_MPRA_mEB_series_mBC_repB_d18_RNA

description: Bulk MPRA

value: Day 18


title: bulk_MPRA_mEB_series_mBC_repB_d21_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d10_RNA

description: Bulk MPRA

value: Day 10


title: bulk_MPRA_mEB_series_mBC_repB_d02_RNA

description: Bulk MPRA

value: Day 02


title: bulk_MPRA_mEB_series_mBC_repB_d14_RNA

description: Bulk MPRA

value: Day 14


title: bulk_MPRA_mEB_series_mBC_repB_d06_RNA

description: Bulk MPRA

value: Day 06


title: bulk_MPRA_mEB_series_mBC_repB_d21_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d16_DNA_extract_lib_rep

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d20_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d14_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d18_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d06_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d10_DNA

description: Bulk MPRA


title: liver_mono_WT_1

type: RNA

city: Braunschweig

first: Andreas

last: Jeron

state: Nierdersachsen

address: Inhoffenstraße 7

department: Viral Immunology

country: Germany

institute: Helmholtz Center for Infection Research

description: Sorted Ly6C-high monocytes from livers of Lm infected wildtype mice on day 3 post infection

hyb_protocol: Hybridization, blocking, staining, washing, was performed acording to Mouse Clariom S microarray manufacturer's recommendations.

scan_protocol: Scanning was performed acording to Mouse Clariom S microarray manufacturer's recommendations.

data_processing: Data were processed with Transcriptome Analysis Console 4.0 (Thermo Fischer) using SST-RMA algorithm.

label: biotin

label_protocol: Labeling was performed acording to Mouse Clariom S microarray manufacturer's recommendations.

extract_protocol: RNA from sorted Ly6C-high monocytes was isolated and purified using the RNeasy Mini Kit (Qiagen) and the Rnase-free DNase kit (Qiagen) according to manufacturer recommendations.

value: wildtype

tag: infection

value: LM

value: 3 Days post LM-infection

value: Ly6C-high monocytes


title: liver_mono_WT_2

source_name: Sorted Ly6C-high monocytes from livers of Lm infected wildtype mice on day 3 post infection


title: bulk_MPRA_mEB_series_mBC_repB_d02_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d20_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d16_RNA_lib_rep

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d21_RNA

description: Bulk MPRA


title: liver_mono_KO_2

description: Sorted Ly6C-high monocytes from livers of Lm infected Nfkbid knockout mice on day 3 post infection

value: Nfkbid knockout


title: bulk_MPRA_mEB_series_mBC_repA_d18_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d14_RNA

description: Bulk MPRA


title: liver_mono_KO_1

source_name: Sorted Ly6C-high monocytes from livers of Lm infected Nfkbid knockout mice on day 3 post infection


title: bulk_MPRA_mEB_series_mBC_repA_d10_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d16_DNA_lib_rep

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d02_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d06_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d21_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d20_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d18_DNA

description: Bulk MPRA


title: sc_rep_mEB_series_mBC_pdT_rep2B_lane2

description:
10X Genomics (custom mBC library)
mBC_poly_dT_counts_sc_rep_mEB_series.txt
assigned_oBC_CRE_mBC_joined_counts_sc_rep_mEB_series.txt


title: sc_rep_mEB_series_mBC_pdT_rep2B_lane1


title: bulk_MPRA_mEB_series_mBC_repA_d10_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d02_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d14_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d06_DNA

description: Bulk MPRA


title: sc_rep_mEB_series_mBC_pdT_repB_lane2


title: sc_rep_mEB_series_GEx_rep2B_lane1

description:
10X Genomics (Gene expression, 3' end)
GEx_obj_sc_rep_mEB_series.RDS
assigned_oBC_CRE_mBC_joined_counts_sc_rep_mEB_series.txt


title: sc_rep_mEB_series_oBC_CS1_rep2B_lane2


title: sc_rep_mEB_series_GEx_rep2B_lane2


title: sc_rep_mEB_series_mBC_pdT_repB_lane1


title: sc_rep_mEB_series_mBC_pdT_repA_lane2


title: sc_rep_mEB_series_mBC_pdT_repA_lane1


title: sc_rep_mEB_series_oBC_CS1_repB_lane2


title: sc_rep_mEB_series_oBC_CS1_repA_lane1


title: sc_rep_mEB_series_GEx_repB_lane2


title: sc_rep_mEB_series_oBC_CS1_repA_lane2


title: sc_rep_mEB_series_GEx_repB_lane1


title: sc_rep_mEB_series_oBC_CS1_repB_lane1


title: sc_rep_mEB_series_GEx_repA_lane2


title: sc_rep_mEB_series_GEx_repA_lane1


title: bulk_MPRA_mEB_series_mBC_rep2B1_d20_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d16_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d16_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d18_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d18_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d20_RNA

description: Bulk MPRA


title: sc_rep_mEB_series_oBC_CS1_rep2B_lane1


title: bulk_MPRA_mEB_series_mBC_rep2B1_d12_RNA

description: Bulk MPRA

value: Day 12


title: bulk_MPRA_mEB_series_mBC_rep2B1_d04_RNA

description: Bulk MPRA

value: WD44 (mouse embryonic stem cells)

value: Day 04


title: bulk_MPRA_mEB_series_mBC_rep2B1_d00_RNA

description: Bulk MPRA

value: Mouse embryoid bodies

value: monoclonal CRISPRi (dCAS9-KRAB) cell line, high MOI (polyclonal) single-cell reporter cassette (89% devCRE library + 10% promoter series + 1% EEF1A1-mCherry) delivered by piggyBac

value: Day 00


title: bulk_MPRA_mEB_series_mBC_rep2B2_d08_RNA

description: Bulk MPRA

value: Day 08


title: bulk_MPRA_mEB_series_mBC_rep2B1_d18_DNA

description: Bulk MPRA

value: Day 18


title: bulk_MPRA_mEB_series_mBC_rep2B1_d08_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d12_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d20_DNA

description: Bulk MPRA

value: Day 20


title: bulk_MPRA_mEB_series_mBC_rep2B1_d16_DNA

description: Bulk MPRA

value: Day 16


title: bulk_MPRA_mEB_series_mBC_rep2B2_d18_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d16_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d12_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d08_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d06_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d12_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d08_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d04_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d00_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d16_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d00_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d16_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d12_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d12_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d20_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d08_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B2_d06_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d04_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d08_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d12_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d16_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d04_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d00_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d04_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d08_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d12_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d08_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d16_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d04_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d00_RNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_rep2B1_d00_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repA_d00_DNA

description: Bulk MPRA


title: bulk_MPRA_mEB_series_mBC_repB_d00_DNA

description: Bulk MPRA


title: ZmPMP3g overexpressing Ye478 treated by moderate drought and ABA rep 3

city: Nanning

first: Ling

last: Lei

state: Guangxi

address: No.100, Daxue East Road

department: College of Life Science and Technology

institute: Guangxi University

data_processing:
Illumina Casava2.19 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm10 whole genome using hisat2 v2.0.5 with parameters hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol from Trapnell,Cole et al., Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol, 2010. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Supplementary files format and content: tab-delimited text file includes readcount values for each Sample

organism: Zea mays

source_name: The second fully-expanded leaves down from the top of plants

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: developmental stage

value: V5 (the fifth leaf stage)

value: leaf

value: ZmPMP3g overexpressing

tag: treatment

value: moderate drought and ABA


title: ZmPMP3g overexpressing Ye478 under control and ABA rep 3

value: control and ABA


title: ZmPMP3g overexpressing Ye478 treated by moderate drought and ABA rep 2


title: ZmPMP3g overexpressing Ye478 under control rep 2

value: control


title: ZmPMP3g overexpressing Ye478 under control and ABA rep 1


title: ZmPMP3g overexpressing Ye478 under control and ABA rep 2


title: ZmPMP3g overexpressing Ye478 treated by moderate drought and ABA rep 1


title: ZmPMP3g overexpressing Ye478 treated by moderate drought rep 2

value: moderate drought


title: Wild type Ye478 under control rep 3

value: Wild type


title: ZmPMP3g overexpressing Ye478 treated by moderate drought rep 3


title: ZmPMP3g overexpressing Ye478 treated by moderate drought rep 1


title: ZmPMP3g overexpressing Ye478 under control rep 1


title: Wild type Ye478 under control and ABA rep 3


title: Wild type Ye478 under control and ABA rep 2


title: Wild type Ye478 under control rep 2


title: Wild type Ye478 under control and ABA rep 1


title: Wild type Ye478 treated by moderate drought and ABA rep 3


title: Wild type Ye478 under control rep 1


title: Wild type Ye478 treated by moderate drought and ABA rep 2


title: Wild type Ye478 treated by moderate drought rep 2


title: sc_rep_promoter_series_mBC_CS2_repB2

description:
10X Genomics (custom mBC library)
mBC_CS2_counts_sc_rep_promoter_series.txt

data_processing:
GEx:
Data was converted to fastq using bcl2fastq, and fastqs were minimally processed (trimming read 1 to 28 cycles with seqtk, files renamed) to be compatible with cellranger (version 6.0.1, 10x Genomics), which was run using reference GRCh38-2020-A. Each CellRanger count output was processed with Seurat. Briefly, cell barcodes were filtered to those with >700 gene expression RNA UMIs, and between 2 and 15% mitochondrial UMI fraction. This led to 5787, 4278, and 3834, cell barcodes across the replicates A, B1, and B2. 10x data was normalized, scaled and clustered using standard commands (NormalizeData with LogNormalize method, finding 1000 top variable features with FindVariableFeatures, scaling with ScaleData over all genes, RunPCA and retaining top 50 principal components [PCs] calculated on the identified variable features, FindNeighbors on the top PCs, FindClusters with 0.1 resolution, and RunUMAP with n.neighbors of 20 and using the top PCs as input features). The UMAP revealed three clear clusters, hypothesized to correspond to the three cell lines profiled. Replicates B1 and B2 also displayed an intermediate cluster, likely as a result of the lane partial wetting failure, found to share marker genes from the focal neighboring clusters, which was excluded as plausibly composed of doublets. To confirm the cellular identity of each cluster, in addition to assessment from canonical marker genes (e.g., HBG1/2 in K562, ALB in HepG2), we compared the pseudo-bulked expression (mean across UMI counts for each gene) to bulk expression quantification in the three lines (as assessed from the average of stranded bulk RNA-seq ENCODE83 datasets in K562 and HepG2, and in HEK293), finding unambiguous correspondence of each clusters to a single line (average log-transformed R^2=0.72 for match, vs. 0.39 for non-match).
Following preliminary filtering described above, cell barcodes corresponding to doublets were removed by two methods. First, each large cluster was further sub-clustered using the same method as above, revealing focal subclusters which shared marker genes from large neighboring clusters, and usually had nearly 2-fold more total RNA UMIs. Cell barcodes contained in these clusters were excluded as doublet from further analysis. Second, scrublet was run on the filtered cell barcode set (>700 RNA UMIs, 2 to 15% mitochondrial RNAs), and a doublet score threshold of 0.25 was selected for filtration based on the separation of the bimodal peaks in the simulated score distribution. Cells either belonging to doublet subclusters or having a scrublet doublet score > 0.25 (we observed high concordance between the two approaches) were filtered out. Finally, cells with anomalously high gene expression UMI (>4000) or anomalously high multiplicity of integration (>100), also likely doublets, were removed, leaving 5505 high confidence cells for replicate A (K562: 2184, HEK293T: 2090, HepG2: 1231), 3533 for replicate B1 (K562: 1303, HEK293T: 1238, HepG2: 992), and 3172 for replicate B2 (K562: 1298, HEK293T: 1056, HepG2: 818).
mBC:
Data was converted to fastq using bcl2fastq, and fastqs were minimally processed (trimming read 1 to 28 cycles and read 2 to 22 cycles with seqtk, files renamed) to be compatible with cellranger (version 6.0.1, 10x Genomics), which was run to perform error correction on cell barcodes. The resulting position sorted bam files were then parsed for the mBC reads as follows using a custom python script. Reads aligning to the reference genome or without either corrected cell barcode or UMI (tags CB and UB in the bam file) were discarded. Only reads with the exact expected 7 nt sequence (TCGACAA) downstream of the mBC (positions 16 to 22) were retained. List of all UMIs corresponding to a cell barcode and mBC pair were stored, discarding chimeric UMIs (taken to be UMIs for which the proportion of reads associated to a given mBC vs all other mBC in the specified cell barcode falls below 0.2). mBC comprised of all Gs (empty read) were discarded. Finally, the UMI count was error corrected as follows. For each given mBC and cell barcode, the Hamming distance between all UMIs was calculated, a graph created by connecting UMIs that were a Hamming distance ≤ 1, and the resulting the number of connected components in the graph was taken as the error-corrected UMI count for a given cell barcode-mBC pair. These error corrected UMI counts were taken as the per single-cell quantification of the reporter mRNA expression (see below for a normalization strategy to correct for gene expression UMIs). Given that cell barcodes derived from capture sequence vs. poly-dT reverse transcription primer are different (bases 8 and 9 reverse complemented) on the same bead (and not error corrected by cellranger in our application), we converted the CS2 cell barcode to its poly-dT counterpart to enable matching across the different libraries.
oBC:
oBC libraries were processed in an entirely analogous way to the strategy for mBC, with the following modifications: two sequencing runs were combined in a single fastq prior to processing, read 2 were trimmed to 23 cycles, and only reads with the GCTTTAA (constant region after the oBC) at positions 17 to 23 were retained. The number of UMIs per oBC per cell barcode was also taken as the error corrected (1 Hamming distance) count and our measure of oBC expression in single cells (see below for a normalization strategy to correct for gene expression UMIs). Similarly to the CS2 mBC data above, we again converted the CS1 cell barcode to poly-dT cell barcodes.
Supplementary files format and content: GEx_obj_sc_rep_promoter_series: Seurat object of quality filtered cells.
Supplementary files format and content: assigned_oBC_CRE_mBC_joined_counts_sc_rep_promoter_series: Final assigned joined cell-oBC-CRE_mBC table. Restricting to oBC with >11 UMI counts, and to uniquely matchable oBC-promoter-mBC triplets. column 1: cell barcode; column 2:  replicate ID; column 3: oBC; column 4: mBC; column 5: CRE class (all promoters here); column 6: CRE identity; column 7: read counts oBC; column 8: UMI counts oBC; column 9: read counts mBC; column 10: UMI counts mBC
Supplementary files format and content: mBC_CS2_counts_sc_rep_promoter_series: raw count table (restricted to mBC from the list determined in the subassembly) per mBC per cell (CS2 captured). Column 1: cell barcode; column 2: replicate ID; column 3: mBC; column 4: read counts; column 5: UMI counts; column 6: capture modality.
Supplementary files format and content: mBC_poly_dT_counts_sc_rep_promoter_series: raw count table (restricted to mBC from the list determined in the subassembly) per mBC per cell (poly-dT captured). Column 1: cell barcode; column 2: replicate ID; column 3: mBC; column 4: read counts; column 5: UMI counts; column 6: capture modality.
Supplementary files format and content: oBC_counts_sc_rep_promoter_series: raw count table (restricted to oBC from the list determined in the subassembly and UMI count >1 to decrease file size) per oBC per cell (CS1 captured). Column 1: cell barcode; column 2: replicate ID; column 3: oBC; column 4: read counts

source_name: Mix of K562, HEK293T, HepG2

growth_protocol:
K562 cells were grown in RPMI 1640 medium (ThermoFisher, cat. num. 11875119), supplemented with 10% FBS and 1x Penicillin/streptomycin (ThermoFisher, cat. num. 15140122). HepG2 and HEK293T cells were grown in DMEM (ThermoFisher, cat. num. 10313021) with 10% FBS and 1x Penicillin/streptomycin. Cells were kept at 37C and 5% CO2, and passaged every two days (K562, HEK293T) or when cells reached confluency (HepG2, typically every three days), except for clonal expansion.
All cells were transfected in mid-exponential phase. K562 cells were transfected using MaxCyte electroporation following manufacturer’s protocol (1.5 M cells, with 15 ug reporter plasmid mix (see above), 0.5 ug super piggybac transposase (SBI) in 50 uL volume). Two replicates of 1 M of HepG2 and HEK293 cells were transfected using lipofectamine 2000 (ThermoFisher) with 4 ug of reporter plasmid mix and 0.2 ug super PiggyBac transposase (SBI). Medium was changed the next day, and cells passaged as usual thereafter. After 5 days, cells were put on puromycin selection, and grown for an additional 10 days to allow complete dilution of the non integrated plasmids. At 15 days post-transfection, cells were bottlenecked to an estimated 250 and 500 starting in clones in multiple replicates, and the populations were expanded before performing bulk vs. single-cell experiments on mid-exponential cultures.

extract_protocol:
The bulk vs. single-cell quantification experiment was performed in two replicates. The first replicate (replicate A) with populations bottlenecked at an expected 250 clones, and the second replicate (replicate B) with populations bottlenecked at an expected 500 clones. For each replicate, at the same time, cells from each line were (1) harvested separately and methanol fixed for bulk quantification, and (2) prepared as single cell suspension, hand-mixed at an expected 1:1 ratio, and profiled for single-cell transcriptomics. Briefly, for the bulk methanol fixation, K562 cells (and HEK293 and HepG2 cells following lifting off plate with 0.05% trypsin) were washed once with ice cold PBS, and resuspended in 80% ice cold methanol, to a concentration of 1 M/mL, and placed at -80C until further processing. For single-cell processing, cells were washed twice with PBS+BSA (0.04%) and diluted to 1000 cells/uL. Cell dilutions were mixed at estimated equal proportion and loaded at expected 10k cells total on the 10x Chromium platform following manufacturer’s protocol (Single Cell 3’ v3.1 with feature barcoding, 10x Genomics), as one lane per replicate. Replicate B showed good emulsion despite having some evidence of a partial wetting failure. Sample processing otherwise proceeded similarly between the two replicates.
For single-cell reporters, three libraries are generated: the standard 3’ gene expression library from 10x (GEx), and two custom derived libraries, one for each reporter RNA (oBC and mBC), obtained from nested PCRs from the amplified cDNA. Briefly, Single-cell library preparation proceeded following the manufacturer's protocol (v3.1 manual CG000205 Rev D, 10x Genomics), with some modifications.
First, one of the replicate’s cDNA (replicate B) was split in two equal halves (and brought to same final volume with elution solution 1) after GEM RT cleanup (step 2.1.s) prior to cDNA amplification to allow for a direct comparison the UMIs captured with different enrichment strategy (hereafter replicate B1 and B2). For cDNA amplification, primers specific to the mBC (oSR38) and oBC (o246) reporter transcripts were spiked-in the reaction (similar to TAP-seq) at final concentration of 0.5 uM to boost UMI capture for replicates A and B1 (but not for replicate B2, to allow direct comparison with replicate B1). Following cDNA amplification, both the bead and supernatant derived material (steps 2.3Ax and 2.3Bxiv respectively) were saved for downstream processing.
Gene expression libraries for all replicates were prepared following the manufacturer’s protocol from 25% of the bead fraction amplified cDNA.
oBC enriched libraries were prepared as follows. For replicate B2 (no primer spiked in), a first outer PCR1 was performed using 25% of the supernatant amplified cDNA with primers oSR40+o246 using Kapa Robust (Roche) and tracking with qPCR until the inflection point (50 uL 2x master mix, 12.5 uL supernatant cDNA, 5 μL 10 μM o246, 5 μL 10 μM oSR40, 0.5 uL SYBr green, and water to 100 μL; run parameters: 3 min at 95C, and cycles 20 s at 95C, 20 s at 60C, 20 s at 72C). Amplicons were cleaned up with 1.75x ampure, and 1/10 of the eluate was carried to the inner PCR with the remaining replicates. For replicates A and B1, the outer PCR was performed during the cDNA amplification via the spiked-in primer, and 25% of the supernatant amplified cDNA was taken as input. Semi-nested inner PCR was performed with primers NextP5_index1 and indexed primers o425-o427, with the same parameters as PCR1 and stopped before the inflection point. Libraries were purified by 1.5x ampure.
As a result of our Pol II reporter construct having a capture sequence (CS2) downstream of the mBC, reporter mRNAs can be captured from both the poly-dT and CS2 reverse transcription primers on the 10x beads. To systematically compare capture efficiency resulting from the two types of primers, two different libraries were generated (poly-dT captured, and CS2 captured). For poly-dT captured libraries, similar to oBC libraries, we first performed outer PCR on replicate B2 (no spiked-in primers in cDNA amplification) using primers oSR38+o207, using the same PCR conditions as for oBC except for an elongation time of 50 s and an anneal temperature of 65C. 25% of the bead-derived amplified cDNA was also used as template. Following 1x ampure clean up, 10% of the eluate was taken for PCR2. PCR2 was performed on all replicates (directly using 25% of the bead-derived amplified cDNA for replicates A and B1) using primers o324+o495 and the same parameters as PCR1, tracking by qPCR and purifying by 1x ampure. A final PCR was performed to index amplicons with primers o076 and indexed primers (o496-o498), and the resulting amplicons purified by 1x ampure. The CS2 libraries were prepared entirely analogously to poly-dT captured libraries, except with the following primers: PCR1 for replicate B2 (SR38+SR40), PCR2 all replicates (o529+oSR40), PCR3 all replicates (NextP5_index1+ indexed primers o530-o532).
Read structure:
GEx: read 1, cell barcode UMI (no custom primer, 66 cycles); index 1, library index (no custom primer, 10 cycles); read 2, transcriptome (no custom primer, 76 cycles)
oBC: read 1, cell barcode UMI (no custom primer, 66 cycles); index 1,  library index (primer o432, 10 cycles); read 2, oBC (primer o433, 76 cycles)
oBC (re-seq): read 1, cell barcode UMI (no custom primer, 34 cycles); index 1, library index (primer o432, 10 cycles); read 2 oBC (primer o433, 38 cycles)
mBC (poly-dT): read 1, cell barcode UMI (no custom primer, 66 cycles); index 1,  library index (primer o494, 10 cycles); read 2, mBC (primer o334, 76 cycles)
mBC (CS2): read 1, cell barcode-UMI (no custom primer, 30 cycles); index 1, library index, (primer o534, 15 cycles); read 2, mBC (primer o334, 18 cycles)

value: Mix of K562, HEK293T, HepG2

value: Mix

value: Polyclonal pools of cells at high MOI integration via piggyBac of single-cell reporters with promoter series. Cellular pool bottlenecked to a few hundred clones, and mixed by hand prior to single-cell sequencing


title: Wild type Ye478 treated by moderate drought rep 3


title: Wild type Ye478 treated by moderate drought rep 1


title: ZmPMP3g overexpressing Ye478 under control rep 3


title: sc_rep_promoter_series_mBC_CS2_repB1


title: sc_rep_promoter_series_oBC_CS1_repB1_reseq

description:
10X Genomics (custom oBC library)
oBC_counts_sc_rep_promoter_series.txt
assigned_oBC_CRE_mBC_joined_counts_sc_rep_promoter_series.txt


title: sc_rep_promoter_series_oBC_CS1_repB2_reseq


title: sc_rep_promoter_series_mBC_CS2_repA


title: sc_rep_promoter_series_oBC_CS1_repA_reseq


title: sc_rep_promoter_series_oBC_CS1_repB2


title: sc_rep_promoter_series_oBC_CS1_repB1


title: sc_rep_promoter_series_mBC_poly_dT_repB2

description:
10X Genomics (custom mBC library)
mBC_poly_dT_counts_sc_rep_promoter_series.txt
assigned_oBC_CRE_mBC_joined_counts_sc_rep_promoter_series.txt


title: sc_rep_promoter_series_mBC_poly_dT_repB1


title: sc_rep_promoter_series_oBC_CS1_repA


title: bulk_MPRA_promoter_series_mBC_DNA_HepG2_repA

description: Bulk MPRA

data_processing:
Sequencing data was demultiplexed using bcl2fastq. Raw fastq files were processed first by trimming unnecessary cycles from the 3’ end (13 cycles from read 1, 4 cycles from read 2, 9 cycles from index 1) using seqtk (https://github.com/lh3/seqtk). Forward and reverse mBC reads were joined and error corrected with PEAR (options -v 15 -m 15 -n 15 -t 15). Using custom python and R scripts, successfully assembled barcode reads were combined with UMI reads, mBC/UMI pairs were counted, and the read counts and UMI count per mBC was determined. The read and UMI counts for the mBC present in the reporter pool (determined a priori, see section on reporter cloning and subassembly) were collected for downstream analysis and comparison to single-cell quantification.
Assembly: N/A
Supplementary files format and content: bulk_MPRA_counts_promoter_series: summary count tables for bulk MPRA data (human cell lines processed separately, promoter series). Column 1: mBC; column 2: CRE identity (one of five promoters in this experiment); column 3: CRE class (all promoters for this experiment); column 4: replicate ID; column 5: cell line; column 6: DNA pseudo UMI counts; column 7: RNA pseudo UMI counts; column 8: DNA read counts; column 9 RNA read counts. mBC with NA the counts were not detected in the respect experiments (0)

extract_protocol:
The bulk vs. single-cell quantification experiment was performed in two replicates. The first replicate (replicate A) with populations bottlenecked at an expected 250 clones, and the second replicate (replicate B) with populations bottlenecked at an expected 500 clones. For each replicate, at the same time, cells from each line were (1) harvested separately and methanol fixed for bulk quantification, and (2) prepared as single cell suspension, hand-mixed at an expected 1:1 ratio, and profiled for single-cell transcriptomics. Briefly, for the bulk methanol fixation, K562 cells (and HEK293 and HepG2 cells following lifting off plate with 0.05% trypsin) were washed once with ice cold PBS, and resuspended in 80% ice cold methanol, to a concentration of 1 M/mL, and placed at -80C until further processing.
Genomic DNA was extracted from methanol fixed cells using the DNeasy kit (Qiagen), and RNA was extracted from cells using TRIzol LS (Thermo Fisher), following manufacturer’s instructions in both cases.
Amplicon libraries from DNA were generated in two steps of PCR amplification with Kapa HiFi (Roche). 0.5-1 ug of genomic DNA input was used. For low-cycle number PCR1, gDNA was mixed with 50 μL 2⨉ Kapa HiFi master mix, 5 μL 10 μM o039, 5 μL 10 μM o358, and water to 100 μL. Cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Primer o358 contains 10 random Ns to serve as a pseudo-UMI (hereafter referred to as UMIs for brevity) to correct for PCR jackpotting. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. Illumina adapters and sequencing indices were appended through PCR2, with 4 μL of the eluate from PCR1 taken as input, and 25 μL 2⨉ Kapa HiFi master mix, 0.25 μL 100⨉ SYBr green, 2.5 μL 10 μM o077, 2.5 μL 10 μM indexed primers (o359-o364), and water to 50 μL. Libraries were amplified with tracking by qPCR with: 1 min at 95C, and cycles up to the qPCR inflection point: 20 s at 98C, 20 s at 60C, 30 s at 72C. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Amplicons libraries for RNA were obtained by first DNase treating RNA (5 μg RNA, 2 μL TURBO DNase [Thermo Fisher], 2 μL 10⨉ buffer, and water to 20 μL, incubated at 37C for 30 min, cleaned up with RNA clean & concentrator [Zymo Research], and eluted in 11 Tris 7 10 mM), and taking 1 μg of DNase treated RNA to reverse transcription. Briefly, 2 μL (500 ng/μL) RNA was mixed with 2 μL 1 μM o358, incubated at 65C for 5 min, and placed on ice. 15 μL of reverse transcription master mix was then added (4 μL 5⨉ FS buffer, 1 μL 0.1 M DTT, 1 μL 10 mM dNTP mix, 8 μL water, 1 μL SSIII [Thermo Fisher]), and the reaction incubated at 55C for 60 min, followed by 70C for 15 min. Half of the reverse transcription reaction was then directly amplified for PCR1 (37.5 2⨉ Kapa HiFi master mix, 3.75 μL o039 10 uM, 3.75 μL o077 10 uM, water to 75 μL), with cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1⨉, and eluted in 20 μL of 10 mM Tris 8. PCR2 proceeded as for libraries prepared from genomic DNA, with o077 and indexing primers (o365, o366, o437-o440), and reactions were stopped at inflexion point from qPCR tracking. Libraries were then cleaned up with Ampure XP beads at 1⨉.
Read structure: read1, mBC forward (28 cycles, primer o369); index1, UMI (19 cycles, primer o435); read2, mBC reverse (19 cycles, primer o371); index2, library index (10 cycles, primer o370)
Bulk MPRA

value: Hepatocellular carcinoma

value: high MOI integration via piggyBac of single-cell reporters with promoter series. Cellular pool bottlenecked to a few hundred clones


title: bulk_MPRA_promoter_series_mBC_RNA_HepG2_repA

description: Bulk MPRA


title: sc_rep_promoter_series_GEx_repB2

description:
10X Genomics (Gene expression, 3' end)
GEx_obj_sc_rep_promoter_series.RDS
assigned_oBC_CRE_mBC_joined_counts_sc_rep_promoter_series.txt


title: sc_rep_promoter_series_GEx_repA


title: sc_rep_promoter_series_mBC_poly_dT_repA


title: bulk_MPRA_promoter_series_mBC_DNA_HepG2_repB

description: Bulk MPRA


title: bulk_MPRA_promoter_series_mBC_DNA_HEK293T_repB

description: Bulk MPRA

value: Embryonic kidney


title: Wild type Ye478 treated by moderate drought and ABA rep 1


title: bulk_MPRA_promoter_series_mBC_DNA_K562_repB

description: Bulk MPRA

value: Erythroleukemia


title: bulk_MPRA_promoter_series_mBC_DNA_HEK293T_repA

description: Bulk MPRA


title: bulk_MPRA_promoter_series_mBC_DNA_K562_repA

description: Bulk MPRA


title: bulk_MPRA_promoter_series_mBC_RNA_HEK293T_repA

description: Bulk MPRA


title: bulk_MPRA_promoter_series_mBC_RNA_K562_repA

description: Bulk MPRA


title: bulk_MPRA_PolIII_linBC_vs_oBC_DNA_rep2

description:
Bulk MPRA
bulk_MPRA_counts_PolIII_oBC_vs_linBC.txt

data_processing:
Sequencing data was demultiplexed using bcl2fastq. Raw fastq files were processed first by trimming unnecessary cycles from the 3’ end (9 cycles from read 1, 4 cycles from read 2) using seqtk (https://github.com/lh3/seqtk). Forward and reverse barcode reads were joined and error corrected with PEAR (options -v 16 -m 16 -n 16 -t 16). Using custom python and R scripts, successfully assembled barcode reads were combined with UMI reads, barcode/UMI pairs were counted, and the read counts and UMI count per barcode was determined. These barcode count files served as the processed inputs for downstream analysis.
For downstream processing, the identity of barcodes present in each transfected plasmid library (p053 and p054) was first determined by inspecting the distribution of barcode UMI counts from separate libraries directly prepared from the respective plasmids. The count distribution displayed clear bimodal nature, and barcodes in the high count mode (>9 UMIs for p053, >5 UMIs for p054) were retained as valid. Following removal of barcodes present in both libraries (34 out of 193705), we were left with a list of barcodes for expression analysis (59.0k for p053, 134.6k for p054).
For quantifying steady-state expression of linear and circular barcodes, we tallied the UMI counts for all valid barcodes for genomic DNA and RNA derived libraries. DNA UMI counts were well correlated from genomic DNA to plasmid (log-transformed R2 comparison of BC UMI= 0.42, and 0.43 for respectively replicates 1 and 2). Steady-state expression (referred to as “activity”) was defined as the normalized RNA UMI counts over the normalized DNA UMI counts, with normalized UMI counts defined as UMI counts over all UMIs mapping to valid barcodes in the respective libraries. For BC well represented in the library (>50 DNA UMI counts), the activity was >150-fold higher for Tornado barcodes compared to linear barcodes (median activity fold-change 162⨉ in rep1 and 186⨉ in rep2).
Assembly: N/A
Supplementary files format and content: bulk_MPRA_counts_PolIII_oBC_vs_linBC: read and UMI counts for each barcode to the two biological replicates. Column 1: biological replicate id; column 2: Pol III barcode; column 3: barcode type (linear linBC or circular oBC); column 4: UMI counts DNA; column 5: read counts DNA; column 6: UMI counts RNA; column 7: read counts RNA.
Supplementary files format and content: bulk_MPRA_PolIII_BC_list: list of barcodes passing read cutoff thresholds for the mapping of the barcode type (circular barcodes from plasmid p053, linear barcodes from plasmid p054). Column 1: Pol III barcode; column 2: UMI count; column 3: read counts; column 4: corresponding plasmid library; column 6: barcode type.

growth_protocol:
K562 cells were grown in RPMI 1640 medium (ThermoFisher, cat. num. 11875119), supplemented with 10% FBS and 1x Penicillin/streptomycin (ThermoFisher, cat. num. 15140122).
2.75 μL each of plasmids libraries p053 and p054 were mixed to 0.6 μL (0.3 μg) of SBI super piggyBac, and transfected 2.5 M of exponentially growing K562 cells in duplicates using a Nucleofector following manufacturer’s protocol for K562 (kit V4XC-2024, Lonza BioResearch) in duplicates. After two weeks of exponential growth with ⅕ split every two days to allow for dilution of unintegrated plasmids, cells were harvested in exponential phase (<0.75 M/mL), and methanol fixed. Briefly, cells were pelleted at 500 g for 5 min, washed with ice cold PBS to 2 M/mL, pelleted at 500 g for 5 min, resuspended to 15 M/mL, and ice cold methanol was added drop by drop to 80%. Aliquots of 4 M fixed cells were stored at -80C until DNA or RNA extractions.

extract_protocol:
Genomic DNA was extracted from methanol fixed cells using the DNeasy kit (Qiagen), and RNA was extracted from cells using TRIzol LS (Thermo Fisher), following manufacturer’s instructions in both cases.
Amplicon libraries from DNA were generated in two steps of PCR amplification with Kapa HiFi (Roche). For genomic DNA, 500 ng of input was used, and for plasmids (to map barcodes present in both constructs), 3 ng was used. For low-cycle number PCR1, 500 ng of DNA was mixed with 50 μL 2⨉ Kapa HiFi master mix, 5 μL 10 μM o246, 5 μL 10 μM o424, and water to 100 μL. Cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Primer o424 contains 10 random Ns to serve as a pseudo-UMI (hereafter referred to as UMIs for brevity) to correct for PCR jackpotting. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1.75⨉, and eluted in 20 μL of 10 mM Tris 8. Illumina adapters and sequencing indices were appended through PCR2, with 4 μL of the eluate from PCR1 taken as input, and 25 μL 2⨉ Kapa HiFi master mix, 0.25 μL 100⨉ SYBr green, 2.5 μL 10 μM o076, 2.5 μL 10 μM indexed primers (DNA rep1: o501, DNA rep2: o502, plasmid p053: o427, plasmid p054: o504), and water to 50 μL. Libraries were amplified with tracking by qPCR with: 1 min at 95C, and cycles up to the qPCR inflection point (typically 15-17 cycles) of: 20 s at 98C, 20 s at 60C, 30 s at 72C. Libraries were then cleaned up with Ampure XP beads at 1.75⨉.
Amplicons libraries for RNA were obtained by first DNase treating the RNA (5 μg RNA, 2 μL TURBO DNase [Thermo Fisher], 2 μL 10⨉ buffer, and waster to 20 μL, incubated at 37C for 30 min, cleaned up with RNA clean & concentrator [Zymo Research], and eluted in 11 Tris 7 10 mM), and taking 1 μg of DNase treated RNA to reverse transcription. Briefly, 2 μL (500 ng/μL) RNA was mixed with 2 μL 1 μM o424, incubated at 65C for 5 min, and placed on ice. 15 μL of reverse transcription master mix was then added (4 μL 5⨉ FS buffer, 1 μL 0.1 M DTT, 1 μL 10 mM dNTP mix, 8 μL water, 1 μL SSIII [Thermo Fisher]), and the reaction incubated at 55C for 60 min, followed by 70C for 15 min. ¼ of the reverse transcription reaction was then directly amplified for PCR1 (37.5 2⨉ Kapa HiFi master mix, 3.75 μL o246, 3.75 μL o076, water to 75 μL), with cycling parameters: 1 min at 95C, and 4 cycles of: 20 s at 98C, 20 s at 60C, 30 s at 72C, followed by 4C hold. Reactions were cleaned up with Ampure XP beads (Beckman Coulter) at 1.75⨉, and eluted in 20 μL of 10 mM Tris 8. PCR2 proceeded as for libraries prepared from plasmids and genomic DNA, with indexing primers o508 and o509 for replicates 1 and 2 respectively, and reactions stopped at inflexion point from qPCR tracking (cycle 7). Libraries were then cleaned up with Ampure XP beads at 1.75⨉.
Read structure: read1, oBC (25 cycles, primer o431); index1, library index (20 cycles, primer o432); read2, oBC reverse (20 cycles, primer o433); index2, UMI (10 cycles, primer o434)
Bulk MPRA

value: high MOI integration via piggyBac of reporters with either hU6 driven oBC/linBC cassettes.


title: CD7p_LIE_B

city: Ghent

first: Tom

last: Taghon

state: East Flanders

address: Corneel Heymanslaan 10

department: Diagnostic Sciences (GE32)

country: Belgium

institute: Ghent University

data_processing:
The quality of the sequencing was verified with FastQC.
Sequencing reads were trimmed using NGMerge.
Alignment was done using Bowtie2 (v2.2.6) with parameter --very-sensitive using the human hg38 reference genome.
Duplicate reads were removed using SAMTools.
Peak calling was performed with MACS2 (v2.1.2) and the option --no-model.
Significant peaks at an adjusted p-value cut-off of 0.05 were combined into one matrix across all samples, with merging of peaks that show at least 50 percent overlap.
The function SummarizeOverlaps of the GenomicAlignments package in R was used for read counting using this matrix and the BAM files.
DESeq2 was used to detect significantly differential opened chromatin sites between samples, using a design containing replicate and condition.
An adjusted p-value of 0.05 was used to retain significant hits where Homer findMotifsGenome was used to perform motif enrichment analysis.
Genome_build: hg38
Supplementary_files_format_and_content: DESeq2 normalized counts
Supplementary_files_format_and_content: Raw counts

source_name: co-cultured HSPCs

growth_protocol: lin-CD123-CD34+GFP+ transduced HSPCs were sorted and co-cultured with OP9-DLL4 stromal cells to induce T cell differentiation.

extract_protocol:
Tagmented genomic DNA was extracted by using Qiagen MinElute PCR purification kit.
DNA libraries were constructed as described by Corces et al., Nat. Methods (2017).

treatment_protocol: HSPCs transduced with IRF8-ERT2-encoding construct were treated with 50 nM of 4-hydroxytamoxifen at day 0 (seeding) and day 3 post co-culture.

value: cord blood

tag: donor

value: B

tag: condition

value: empty vector control

value: untreated

tag: phenotype


title: CD7p_LIE_C

value: C


title: bulk_MPRA_PolIII_linBC_vs_oBC_DNA_rep1

tag: cell line

tag: genotype


title: CD7p_LIE_A

value: A


title: bulk_MPRA_PolIII_linBC_vs_oBC_RNA_rep2

tag: cell type


title: bulk_MPRA_PolIII_linBC_vs_oBC_RNA_rep1


title: sc_rep_promoter_series_GEx_repB1


title: bulk_MPRA_promoter_series_mBC_RNA_HepG2_repB

description: Bulk MPRA


title: CD7p_IRF8ERT2_C

value: 50 nM of 4-hydroxytamoxifen


title: bulk_MPRA_promoter_series_mBC_RNA_HEK293T_repB

description: Bulk MPRA


title: CD7p_IRF8ERT2_B


title: CD7m_LIE_C


title: CD7p_IRF8ERT2_A


title: CD7m_LIE_A


title: CD7m_IRF8ERT2_C


title: CD7m_LIE_B


title: bulk_MPRA_promoter_series_mBC_RNA_K562_repB

description: Bulk MPRA