title: ChIP-seq_Oct4_15h_rep2

status: Public on Jun 30 2021

type: SRA

city: Göttingen

first: Le

last: Xiong

address: Fassberg 11

department: Molecular Biology

country: Germany

institute: MPI for biophysical chemistry

library_source: genomic

data_processing:
For TT-seq data: Paired-end 42 bp reads were mapped to the mouse genome assembly mm10 using STAR 2.5.3 (Dobin et al., 2013) with the following parameters: outFilterMismatchNmax 2, outFilterMultimapScoreRange 0 and alignIntronMax 500,000. SAMtools (Li et al., 2009) were then used to remove alignments with MAPQ smaller than 7 (-q 7) and only proper pairs (-f 2) were selected. HTSeq-count (Anders et al., 2015) was used to calculate fragment counts for different features. R/Bioconductor package DESeq2 (Love et al., 2014) was used to call differentially expressed mRNAs and eRNAs applying DESeq2’s default size factor normalization. For both cases DESeq2 size factors were calcualted using counts for protein-coding genes. An adjusted P-value of 0.01 was used to identify significantly changed mRNAs or eRNAs by comparing each time point to the 0 hours measurement.  Further data processing was carried out using the R/Bioconductor environment.
For ATAC-seq data: Paired-end 76 bp reads were obtained for each of the samples and Nextera Transposase adapter sequence was removed using (Martin, 2011). Bowtie2 (Langmead and Salzberg, 2012) was used to align paired-end reads to the mouse genome assembly mm10 with the “--local” and “--no-discordant” options. SAMtools (Li et al., 2009) was then used to remove alignments with MAPQ smaller than 7 (-q 7) and only proper pairs (-f 2) were selected. Reads mapped to custom blacklist regions and mitochondria were removed. Two replicates were pooled and MACS2 (Zhang et al., 2008) was used to call chromatin opening peaks with options: -f BAMPE -g mm --broad --broad-cutoff 0.05. Peaks for all time point were then merged to create non-overlapping unified peaks. For quantitative comparison, HTSeq-count (Anders et al., 2015) was used to calculate fragment counts for the non-overlapping unified peaks and DESeq2 was used to call regions with significantly changed chromatin accessibility. For normalization, ATAC-seq peaks overlapping with promoters of protein-coding genes with unchanged mRNA expression were used to calculate DESeq2 size factors. An adjusted P-value of 0.01 was used to identify significantly changed regions by comparing each time point to the 0 hour measurement. Further data processing was carried out using the R/Bioconductor environment.
For ChIP-seq data: Paired-end Oct4 and Sox2 ChIP-seq data processing was done as described for ATAC-seq data. For peak calling, MACS2 was used with the same option for each individual replicate. For Oct4 ChIP-seq data, data was normalized using added D. melanogaster RNA spike-ins. Normalization factors were obtained by dividing the total D. melanogaster spike-ins read counts for each sample by the total read counts of the sample with the lowest read counts. For Sox2 ChIP-seq data, data was normalized using total number of uniquely mapped reads. ChIP-seq coverages were divided by the respective normalization factors. HTSeq-count (Anders et al., 2015) was used to calculate fragment counts for peak features. Reads counts were divided by the respective normalization factors.
Genome_build: mm10
Supplementary_files_format_and_content: Normalized coverage files for TT-seq, ATAC-seq, Oct4 and Sox2 ChIP-seq at each time point are provided in bigwig format. For ChIP-seq data, two replicates are merged.
Supplementary_files_format_and_content: Annotations of transcription unit, eRNA, Oct4-regulated eRNA, Oct4-bound transcribed enhancers, Oct4-sensitive and -insensitive enhancers are provided in gtf format.

molecule: genomic DNA

organism: Mus musculus

source_name: ZHBTc4 mouse embryonic stem cell

growth_protocol: Mouse embryonic stem cells (mESCs) harbouring a doxycycline repressible Pou5f1 transgene (ZHBTc4, Niwa et al.,  2000) were propagated on gelatin-coated plates in equal parts DMEM-F12 (Life Technology, 21331-020) and Neuralbasal (Life Technology, 21103-049) Medium supplemented with 2% Fetal Bovin Serum (Sigma Aldrich, G1393-100 ml), 2% Knockout Serum Replacement Medium (Gibco, 10828-028), 0.04 µg/ml Leukemia Inhibitory Factor (prepared in-house), Pennicilin/Streptomycin (Sigma Aldrich, P4333-100ml), 0.1 mM ß-Mercaptoethanol (Gibco, 31350-010), 0.5x B27 supplement (Life Technology, 12587-010), 0.5x N2 supplement (Gibco, AM9759, 3 µM CHIR99021 (Cayman Chemicals, 13122), 1 µM PD0325901 (Biomol, 103034-25). Cells were passaged using Accutase (Sigma Aldrich, 6964-100ml).

extract_protocol:
ChIP-seq
NEBNext® Ultra™ II DNA Library Prep Kit

treatment_protocol: Pou5f1 expression was abolished by treatment with 1µg/ml doxycycline (Sigma Aldrich D9891-1G) for 3 hours, 6 hours, 9 hours, 12 hours and 15 hours.

tag: cell line

value: ZHBTc4 mouse embryonic stem cell

tag: treatment condition

value: doxycycline (1ug/ml) treatment 15h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_12h_rep1

value: doxycycline (1ug/ml) treatment 12h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_15h_rep1


title: ChIP-seq_Oct4_12h_rep2


title: ChIP-seq_Oct4_3h_rep2

value: doxycycline (1ug/ml) treatment 3h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_6h_rep1

value: doxycycline (1ug/ml) treatment 6h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_0h_rep1

value: untreated control 0h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_6h_rep2


title: ChIP-seq_Oct4_3h_rep1


title: ChIP-seq_Oct4_9h_rep2

value: doxycycline (1ug/ml) treatment 9h, Oct4 antibody (R&D, AF1759)


title: ChIP-seq_Oct4_9h_rep1


title: ATAC-seq_15h_rep2

extract_protocol:
ATAC-seq (Buenrostro et al. 2015)
Nextera Tn5 enzyme-NEBNext High fidelity master mix

value: doxycycline (1ug/ml) treatment 15h


title: ATAC-seq_15h_rep1


title: ATAC-seq_12h_rep2

value: doxycycline (1ug/ml) treatment 12h


title: ATAC-seq_12h_rep1


title: ATAC-seq_3h_rep1

value: doxycycline (1ug/ml) treatment 3h


title: ATAC-seq_3h_rep2


title: ATAC-seq_6h_rep1

value: doxycycline (1ug/ml) treatment 6h


title: ATAC-seq_9h_rep1

value: doxycycline (1ug/ml) treatment 9h


title: ATAC-seq_9h_rep2


title: ATAC-seq_0h_rep2

value: untreated control 0h


title: ATAC-seq_0h_rep1


title: ChIP-seq_Oct4_0h_rep2


title: TT-seq_15h_rep2

description: 4sU-labeled RNA

library_source: transcriptomic

molecule: total RNA

extract_protocol:
TT-seq (Schwalb et al, 2016)
Ovation Universal RNA-seq System (Nugen, 0343)

value: doxycycline (1ug/ml) treatment 15h + 5 min 4sU labeling


title: TT-seq_15h_rep1

description: 4sU-labeled RNA


title: TT-seq_12h_rep2

description: 4sU-labeled RNA

value: doxycycline (1ug/ml) treatment 12h + 5 min 4sU labeling


title: TT-seq_12h_rep1

description: 4sU-labeled RNA


title: TT-seq_9h_rep1

description: 4sU-labeled RNA

value: doxycycline (1ug/ml) treatment 9h  + 5 min 4sU labeling


title: TT-seq_3h_rep2

description: 4sU-labeled RNA

value: doxycycline (1ug/ml) treatment 3h + 5 min 4sU labeling


title: TT-seq_6h_rep2

description: 4sU-labeled RNA

value: ZHBTc4 mouse embryonic stem cell

value: doxycycline (1ug/ml) treatment 6h + 5 min 4sU labeling


title: TT-seq_0h_rep1

description: 4sU-labeled RNA

value: untreated control 0h  + 5 min 4sU labeling


title: TT-seq_9h_rep2

description: 4sU-labeled RNA

value: doxycycline (1ug/ml) treatment 9h + 5 min 4sU labeling


title: NASA-M35 [RNA-Seq]

city: Loma Linda

first: Charles

last: Wang

state: CA

address: 11021 Campus Street

department: Department of Basic Sciences

country: USA

institute: Loma Linda University

description:
stranded, rRNA depletion
CFG525

data_processing:
NextSeq 550 onboard basecalling, fastq files were generated using Illumina bcl2fastq package v1.8.4 with default settings.
RRBS fastq header contained 6 nt index sequence and 6 random nt (N6 NuGen RRBS duplicate information). Adaptors and low quality reads were trimmed with Trim_galore v0.4.5. and reads containing adapter were discarded. Following adaptor and quality trimming, the fastq files were further trimmed with NuGEN custom python script trimRRBSdiversityAdaptCustomers.py. Potential PCR or optical duplicates were removed using NuGEN python script Nudup.py
Trimmed reads was aligned to the mouse genome GRCm38, allowing for 1 mismatch and retaining only uniquely mapped reads. Bam files were generated using Bismark v0.16.334. CpG coverage reports were generated using MethylKit from bam files.
RNA-seq fastq files were trimmed with trimmomatic, aligned to mouse genome with Tophat2, and reads were qunatified with cutfflinks. Gene differential expression was analyzed using cuffdiff with FPKM threshold of 1.
Genome_build: GRCm38
Supplementary_files_format_and_content: Processed methylation data is uploaded as CpG reports in txt format. Process RNA-seq data is uploaded as normalized FPKM count matrix in csv format.

source_name: Control, right retina

extract_protocol:
QIAGEN AllPrep DNA/RNA/miRNA Universal Kit protocol
RRBS: NuGen Ovation RRBS Methyl-Seq protocol with QIAGEN EpiTech Fast DNA Bisulfite Kit for bisulfite conversion. RNA-seq NuGen Universal RNA-seq library protocol for moouse.

tag: condition

value: Ground habitat control

tag: tissue

value: retina

tag: age (week)

tag: Sex

value: female

tag: strain

value: C57 BL/6J


title: NASA-M34 [RNA-Seq]

description:
stranded, rRNA depletion
CFG522


title: NASA-M37 [RNA-Seq]

description:
stranded, rRNA depletion
CFG527


title: TT-seq_3h_rep1

description: 4sU-labeled RNA


title: TT-seq_0h_rep2

description: 4sU-labeled RNA

value: untreated control 0h + 5 min 4sU labeling


title: NASA-M36 [RNA-Seq]

description:
stranded, rRNA depletion
CFG526


title: TT-seq_6h_rep1

description: 4sU-labeled RNA


title: NASA-M32 [RNA-Seq]

description:
stranded, rRNA depletion
CFG520

source_name: Control, left retina


title: ATAC-seq_6h_rep2


title: NASA-M33 [RNA-Seq]

description:
stranded, rRNA depletion
CFG521


title: NASA-M29 [RNA-Seq]

description:
stranded, rRNA depletion
CFG517

source_name: Flight, left retina

value: Spaceflight


title: NASA-M26 [RNA-Seq]

description:
stranded, rRNA depletion
CFG515

source_name: Flight, right retina


title: NASA-M27 [RNA-Seq]

description:
stranded, rRNA depletion
CFG516


title: NASA-M30 [RNA-Seq]

description:
stranded, rRNA depletion
CFG518


title: NASA-M25 [RNA-Seq]

description:
stranded, rRNA depletion
CFG514


title: NASA-M24 [RNA-Seq]

description:
stranded, rRNA depletion
CFG513


title: NASA-M31 [RNA-Seq]

description:
stranded, rRNA depletion
CFG519


title: NASA-M23 [RNA-Seq]

description:
stranded, rRNA depletion
CFG512


title: NASA-M21 [RNA-Seq]

description:
stranded, rRNA depletion
CFG510


title: NASA-M37 [RRBS]


title: NASA-M39 [RRBS]


title: NASA-M36 [RRBS]


title: NASA-M22 [RNA-Seq]

description:
stranded, rRNA depletion
CFG511


title: NASA-M33 [RRBS]


title: NASA-M32 [RRBS]


title: NASA-M31 [RRBS]


title: NASA-M35 [RRBS]


title: NASA-M34 [RRBS]


title: NASA-M26 [RRBS]


title: NASA-M24 [RRBS]


title: NASA-M23 [RRBS]


title: NASA-M22 [RRBS]


title: NASA-M29 [RRBS]


title: NASA-M25 [RRBS]


title: NASA-M27 [RRBS]


title: NASA-M21 [RRBS]


title: RNASeq_14DaysPI rep2

city: Liverpool

first: Mark

middle: J

last: Taylor

address: Pembroke Place

department: Tropical Disease Biology

country: United Kingdom

institute: Liverpool School of Tropical Medicine

description:
Day14-2
Counts-wBm.txt
Counts-Bmy.txt

data_processing:
Basecalls performed using CASAVA version 1.8
FastQ files were trimmed for presence of adapter sequences (Cutadapt version 1.2.1, options "-O 3") and low-quality reads (Sickle version 1.3.3)
Fastq files mapped to the B. malayi genome using the Subjunc alignment program (version 1.5.0), and read counts obtained using FeatureCounts (version 1.5.0)
Unmapped reads then extracted from the BAM files and remapped to the Wolbachia of B. malayi genome using the Subread-Aligner program (version 1.5.0), and read counts obtained using FeatureCounts (version 1.5.0)
ID conversions were then performed for the B. malayi genome using the online bioDBnet tool 'Db2Db' for downstream applications, using as input 'GeneID' and output as 'Kegg Gene ID'
Normalized abundance measurements were then conducted using EdgeR for both organisms and their read counts as calculated previously
Genome_build: GCF_000002995.3_ASM299v2_genomic (Brugia malayi genome)
Genome_build: GCF_000008385.1_ASM838v1_genomic (Wolbachia endosymbiont genome)
Supplementary_files_format_and_content: Tab-delimited text files containing read counts as determined by FeatureCounts for both Brugia malayi and the Wolbachia endosymbiont

organism:
Brugia malayi
Wolbachia endosymbiont strain TRS of Brugia malayi

source_name: Brugia malayi and Wolbachia endosymbiont

growth_protocol: The Brugia malayi nematode life cycle is maintained at the Filariasis Research Reagent Resource Centre, using a combination of mosquito blood feeds and intraperitoneal injections/washes of mongolian gerbil animal models

extract_protocol:
Batches of nematodes were defrosted and RNA extracted using Trizol reagent. Upper aqueous phases were then aspirated out, bound to, washed and eluted using an RNeasy mini kit (Qiagen). Ribosomal RNA species were then depleted via use of Terminator Exonuclease (Epicentre).
Illumina GA-IIx cDNA libraries were then prepared using ScriptSeq v2 library preparation kit (Epicentre), according to manufacturer's instructions.

tag: host developmental stage

tag: time

value: 14 days post-infection

value: whole organism


title: RNASeq_11DaysPI rep2

description:
Day11-2
Counts-wBm.txt
Counts-Bmy.txt

value: 11 days post-infection


title: RNASeq_14DaysPI rep1

description:
Day14-1
Counts-wBm.txt
Counts-Bmy.txt


title: RNASeq_11DaysPI rep1

description:
Day11-1
Counts-wBm.txt
Counts-Bmy.txt


title: RNASeq_7DaysPI rep2

description:
Day7-2
Counts-wBm.txt
Counts-Bmy.txt

value: 7 days post-infection


title: RNASeq_7DaysPI rep3

description:
Day7-3
Counts-wBm.txt
Counts-Bmy.txt


title: RNASeq_7DaysPI rep1

description:
Day7-1
Counts-wBm.txt
Counts-Bmy.txt


title: fnrL mutant, denitrification_shift_2

type: RNA

city: Bunkyo-ku

first: Hiroyuki

last: Arai

state: Tokyo

address: Yayoi 1-1-1

department: Department of Biotechnology

country: Japan

institute: The University of Tokyo

description: This sample is of R. denitrificans OCh114 fnrL mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the second of two biological replicates used in this experiment, each from separate cultures.

hyb_protocol: Hybridization was performed with Hybridization System 4 according to the NimbleGen Arrays User’s Guide for Gene Expression Analysis (Roche NimbleGen).

scan_protocol: Array scanning was performed with MS200 Microarray Scanner according to the NimbleGen Arrays User’s Guide for Gene Expression Analysis (Roche NimbleGen).

data_processing: The raw data (.pair file) was subjected to RMA, quantile normalization, and background correction as implemented in the NimbleScan software 2.5 (Roche NimbleGen).

organism: Roseobacter denitrificans OCh 114

source_name: fnrL mutant, denitrification_shift dark replicate 2

label_protocol: Labeling of double stranded cDNA synthesized with the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen) was performed according to the instructions for NimbleGen Arrays User’s Guide for Gene Expression Analysis by Roche NimbleGen using One-Color DNA Labeling Kit (Roche NimbleGen).

growth_protocol: R. denitrificans strains were cultivated aerobically in glycerol medium supplemented with 20 mM NaNO3 in a baffled Erlenmeyer flask at 30˚C with rotary shaking at 200 rpm. When optical density at 600 nm was 0.4, an aliquot of the culture was moved to a vial. After the vial was sealed with a butyl rubber septum, the headspace of the vial was replaced with argon. Total RNA was extracted after anaerobic cultivation for 3 hours.

extract_protocol: Total RNA was extracted by a hot-phenol method. After contaminated DNA was eliminated by RQ1 DNase (Promega) treatment, RNA was purified by RNeasy mini columns (Qiagen).

treatment_protocol: The culture was mixed with 2 volumes of RNAprotect Bacterial Reagent (Qiagen) before RNA purification.

tag: genotype

value: fnrL mutant

tag: growth condition

value: denitrification_shift dark


title: RNASeq_14DaysPI rep3

description:
Day14-3
Counts-wBm.txt
Counts-Bmy.txt


title: RNASeq_3DaysPI rep3

description:
Day3-3
Counts-wBm.txt
Counts-Bmy.txt

value: 3 days post-infection


title: RNASeq_3DaysPI rep1

description:
Day3-1
Counts-wBm.txt
Counts-Bmy.txt


title: RNASeq_3DaysPI rep2

description:
Day3-2
Counts-wBm.txt
Counts-Bmy.txt


title: fnrL mutant, denitrification_shift_1

description: This sample is of R. denitrificans OCh114 fnrL mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: fnrL mutant, denitrification_shift dark replicate 1


title: dnr1 dnr2 mutant, denitrification_shift_2

description: This sample is of R. denitrificans OCh114 dnr1 dnr2 double mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr1 dnr 2 double mutant, denitrification_shift dark replicate 2

value: dnr1 dnr2 double mutant


title: dnr1 dnr2 mutant, denitrification_shift_1

description: This sample is of R. denitrificans OCh114 dnr1 dnr2 double mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr1 dnr 2 double mutant, denitrification_shift dark replicate 1


title: dnr2 mutant, denitrification_shift_2

description: This sample is of R. denitrificans OCh114 dnr2 mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr2 mutant, denitrification_shift dark replicate 2

value: dnr2 mutant


title: dnr1 mutant, denitrification_shift_2

description: This sample is of R. denitrificans OCh114 dnr1 mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr1 mutant, denitrification_shift dark replicate 2

value: dnr1 mutant


title: fetal end rep3

city: Salt Lake City

first: Robert

middle: Allen

last: McKnight

state: UT

address: Wakara Way

department: Pediatrics

institute: University of Utah

description: 17303X15_190920_A00421_0111_AHFNJFDRXX_S61_L002

data_processing:
De-multiplexed fastq files were generated with Illumina bcl2fastq
Optical duplicates were removed from the Fastq file using BBmap clumpify (version 38.34) and a pixel distance of 12000. Standard Illumina adapter sequences were then removed using Cutadapt (version 1.16) with a minimum matching sequence (option -O) of 6 bp and discarding reads (option -m) below 20 bp.
Processed reads were aligned to the genome with STAR (version 2.7.0f) using Ensembl Ovis aries 3.1 genome and annotation (release 96) with two-pass basic alignment.
Read counts for genes were generated using Subread featureCounts (version 1.6.3) and Ensembl annotation (release 96), assigning reads to the gene with the largest overlap. Read counts were used in differential expression with DESeq2 (version 1.22.2).
Genome_build: Ensembl Oar_v3.1, release 96
Supplementary_files_format_and_content: Tab-delimited text file of gene counts for each sample

organism: Ovis aries

source_name: right lower lung lobe_fetal end

extract_protocol:
Lung tissue was removed, flash frozen on dry ice, and RNA was harvested using Macherey-Nagel NucleoSpin RNA kit. Illumina TruSeq Stranded Total RNA Library Prep Kit.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: animal id

tag: age

tag: treatment

value: right lower lung lobe


title: dnr2 mutant, denitrification_shift_1

description: This sample is of R. denitrificans OCh114 dnr2 mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr2 mutant, denitrification_shift dark replicate 1


title: fetal end rep2

description: 17303X14_190920_A00421_0111_AHFNJFDRXX_S63_L002


title: fetal end rep1

description: 17303X13_190920_A00421_0111_AHFNJFDRXX_S52_L002


title: fetal start rep3

description: 17303X12_190920_A00421_0111_AHFNJFDRXX_S54_L002

source_name: right lower lung lobe_fetal start


title: dnr1 mutant, denitrification_shift_1

description: This sample is of R. denitrificans OCh114 dnr1 mutant cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: dnr1 mutant, denitrification_shift dark replicate 1


title: RNASeq_11DaysPI rep3

description:
Day11-3
Counts-wBm.txt
Counts-Bmy.txt


title: fetal start rep2

description: 17303X11_190920_A00421_0111_AHFNJFDRXX_S56_L002


title: fetal start rep1

description: 17303X10_190920_A00421_0111_AHFNJFDRXX_S51_L002


title: NRS rep3

description: 17303X9_190920_A00421_0111_AHFNJFDRXX_S53_L002

source_name: right lower lung lobe_NRS

value: noninvasive respiratory support


title: microaerobic_IR_1

description: This sample is of R. denitrificans OCh114 cells during growth under the microaerobic condition with near-infrared light irradiation. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, microaerobic near-infrared light irradiation replicate 1

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium in a glass bottle at 30˚C with continuous bubbling of a gas mixture of 2% O2 and 98% N2. The bottle was irradiated with LED near-infrared light (730 nm) at 30 W m-2. Total RNA was extracted when optical density at 600 nm was 0.4-0.42.

value: wild type


title: NRS rep2

description: 17303X8_190920_A00421_0111_AHFNJFDRXX_S55_L002


title: aerobic_IR_2

description: This sample is of R. denitrificans OCh114 cells during growth under the aerobic condition with near-infrared light irradiation. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic near-infrared light irradiation replicate 2

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium in a glass bottle at 30˚C with continuous bubbling of air. The bottle was irradiated with LED near-infrared light (730 nm) at 30 W m-2. Total RNA was extracted when optical density at 600 nm was 0.4-0.42.

value: aerobic near-infrared light irradiation


title: aerobic_blue_1

description: This sample is of R. denitrificans OCh114 cells during growth under the aerobic condition with blue light irradiation. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic blue light irradiation replicate 1

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium in a glass bottle at 30˚C with continuous bubbling of air. The bottle was irradiated with LED blue light (470 nm) at 30 W m-2. Total RNA was extracted when optical density at 600 nm was 0.4-0.42.

value: aerobic blue light irradiation


title: aerobic_blue_2

description: This sample is of R. denitrificans OCh114 cells during growth under the aerobic condition with blue light irradiation. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic blue light irradiation replicate 2


title: denitrification_shift_2

description: This sample is of R. denitrificans OCh114 cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification_shift dark replicate 2

growth_protocol: R. denitrificans OCh114 was cultivated aerobically in glycerol medium supplemented with 20 mM NaNO3 in a baffled Erlenmeyer flask at 30˚C with rotary shaking at 200 rpm. When optical density at 600 nm was 0.4, an aliquot of the culture was moved to a vial. After the vial was sealed with a butyl rubber septum, the headspace of the vial was replaced with argon. Total RNA was extracted after anaerobic cultivation for 3 hours.


title: NRS rep1

description: 17303X7_190920_A00421_0111_AHFNJFDRXX_S57_L002


title: denitrification_IR_2

description: This sample is of R. denitrificans OCh114 cells during growth under the denitrification condition with near-infrared light irradiation. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification near-infrared light irradiation replicate 2

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium supplemented with 20 mM NaNO3 in a side-armed Erlenmeyer flask at 30˚C . The flask was sealed with a silicone plug and a butyl rubber septum and the headspace of the flask was replaced with argon. The flask was irradiated with LED near-infrared light (730 nm) at 30 W m-2. Total RNA was extracted when optical density at 600 nm was 0.2-0.23.


title: denitrification_IR_1

description: This sample is of R. denitrificans OCh114 cells during growth under the denitrification condition with near-infrared light irradiation. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification near-infrared light irradiation replicate 1


title: microaerobic_IR_2

description: This sample is of R. denitrificans OCh114 cells during growth under the microaerobic condition with near-infrared light irradiation. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, microaerobic near-infrared light irradiation replicate 2


title: aerobic_IR_1

description: This sample is of R. denitrificans OCh114 cells during growth under the aerobic condition with near-infrared light irradiation. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic near-infrared light irradiation replicate 1


title: IMV rep3

description: 17303X6_190920_A00421_0111_AHFNJFDRXX_S58_L002

source_name: right lower lung lobe_IMV

value: invasive mechanical ventilation


title: term rep3

description: 17303X3_190920_A00421_0111_AHFNJFDRXX_S62_L002

source_name: right lower lung lobe_term


title: IMV rep2

description: 17303X5_190920_A00421_0111_AHFNJFDRXX_S59_L002


title: IMV rep1

description: 17303X4_190920_A00421_0111_AHFNJFDRXX_S60_L002


title: denitrification_dark_1

description: This sample is of R. denitrificans OCh114 cells during growth under the dark denitrification condition. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification dark replicate 1

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium supplemented with 20 mM NaNO3 in a side-armed Erlenmeyer flask at 30˚C . The flask was sealed with a silicone plug and a butyl rubber septum and the headspace of the flask was replaced with argon. Total RNA was extracted when optical density at 600 nm was 0.2-0.23.

value: denitrification dark


title: denitrification_dark_2

description: This sample is of R. denitrificans OCh114 cells during growth under the dark denitrification condition. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification dark replicate 2


title: denitrification_shift_1

description: This sample is of R. denitrificans OCh114 cells after the growth condition was shifted from dark aerobic to dark denitrification. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, denitrification_shift dark replicate 1


title: term rep2

description: 17303X2_190920_A00421_0111_AHFNJFDRXX_S64_L002


title: 87H

city: Edinburgh

first: Stephen

last: Larcombe

state: Lothian

address: Charlotte Auerbach Road

institute: University of Edinburgh

data_processing:
Lane on the Illumina HiSeq 4000 (Paired-end, 2x150 bp sequencing, generating data from >280 M clusters per lane)
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to the bos taurus genome (UMD3.1)  using bowtie2 for paried end reads with paramters -p 8 -x  -1 pairedendreadsfile1.fastq -2 pariedendreadsfile2.fastq -S
Genome_build: Bos taurus UMD3.1
Supplementary_files_format_and_content: tab-delimited text files include DESeq2 output file containing corrected read abundance on by sample basis as well as calculate group comparisons  including p-value, adjusted p-vlues, log2 fold change and Ensembl and gene IDs

organism: Bos indicus

source_name: T.annulata infected Leukocyte cell line

growth_protocol: The cell-lines were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% FBS, 4mM L-glutamine and 50 µM β-mercaptoethanol at 37°C and harvested during the exponential phase of growth.  The Sahiwal cell-lines were between passages 4-6 and Holstein-Friesian cell-lines were between passages 6-8.

extract_protocol:
Cells were harvested by centrifugation (120 g for 10 minutes) and RNA isolated using Trizol (Thermo Fisher), followed by further purification using the RNeasy mini kit (Qiagen), including an on-column DNAse digestion step, using the suppliers’ protocols.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: breed

value: sahiwal breed

tag: passage

value: harvested passage 4-6


title: 85H


title: 83H


title: 84H


title: 86H


title: microaerobic_dark_2

description: This sample is of R. denitrificans OCh114 cells during growth under the dark microaerobic condition. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, microaerobic dark replicate 2

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium in a glass bottle at 30˚C with continuous bubbling of a gas mixture of 2% O2 and 98% N2. Total RNA was extracted when optical density at 600 nm was 0.4-0.42.

value: microaerobic dark


title: microaerobic_dark_1

description: This sample is of R. denitrificans OCh114 cells during growth under the dark microaerobic condition. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, microaerobic dark replicate 1


title: 82H


title: aerobic_dark_2

description: This sample is of R. denitrificans OCh114 cells during growth under the dark aerobic condition. It is the second of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic dark replicate 2

growth_protocol: R. denitrificans OCh114 was cultivated in glycerol medium in a glass bottle at 30˚C with continuous bubbling of air. Total RNA was extracted when optical density at 600 nm was 0.4-0.42.

value: aerobic dark


title: aerobic_dark_1

description: This sample is of R. denitrificans OCh114 cells during growth under the dark aerobic condition. It is the first of two biological replicates used in this experiment, each from separate cultures.

source_name: R. denitrificans, aerobic dark replicate 1


title: Pot_Pi-GFP_IP_rep1

city: Uppsala

first: Kristian

middle: Erik

last: Persson Hodén

address: Almas allé 5

department: Plant Biology

country: Sweden

institute: Swedish University of Agricultural Sciences

description: Phytophthora infestans,  Solanum tuberosum (Bintje)

data_processing:
Cutadapt, quality trimming (20), read length filtration 18 - 38 nt
Bowtie2 in combination with SAMtools view were used for tRNA and rRNA trimming and potato and P. infestans sRNA filtering to exclude the reads from the other genome for the different datasets, using 0 mismatches allowed per seed against each Fasta filter
SamToFastq converted the files to fastq
Fastx_collapser in the FASTX-toolkit counted the number of St- and Pi-sRNA
ShortStack was used to predict miRNA and summarize sRNA, using strand cutoff 0.5, fold size 1000, dicermin 18 and dicermax 38
Genome_build: Solanom tuberosum genome v4.04
Genome_build: Phytophthora infestans genome (ASM14294v1)
Supplementary_files_format_and_content: Results.txt files from the output of Shortstack including predicted sRNA

organism:
Solanum tuberosum
Phytophthora infestans

source_name: Ago-RNA co-immunoprecipitation

extract_protocol:
Mycelia and infected leaves were harvested and flash frozen in liquid nitrogen. PiAgo1-GFP and control Pi-GFP immunoprecipitations were performed with anti-GFP antibody (Chromotek, gtm-100).
Barcoded sequencing libraries were constructed using the Ion Total RNA-Seq Kit v2 and Ion Xpress barcode primers. Sequencing was performed on the Ion Proton platform using the Ion PI™ Hi-Q™ Sequencing 200 Kit (Thermo Fisher Scientific).

tag: gfp-tagged protein

tag: wild-type background

tag: transformed construct

value: GFP

value: Mycelium, Leaves

tag: potato line

value: Bintje

tag: ip antibody

value: GFP (Chromotek, gtm-100)


title: Pot_PiAgo1-GFP_IP_rep1

description: Phytophthora infestans, Solanum tuberosum (Bintje)

tag: tissue


title: Pot_Pi-GFP_IP_rep3


title: Pot_PiAgo1-GFP_IP_rep3


title: Pot_PiAgo1-GFP_IP_rep2


title: Pot_Pi-GFP_IP_rep2


title: Em organoid_K5_#5_before

city: Chiba

first: Yoshitaka

last: Hippo

state: Chiba

address: 666-2 Nitona-cho, Chuo-ku

institute: Chiba Cancer Center

description:
Em organoid_K5_#5_before
Gene expression of Em organoids with KrasG12D and Trp53 loss before subcutaneous inoculation, #5

hyb_protocol: Gene Expression Hybridization Kit (Agilent) were used for hybridization according to the manufacturer's instructions. After hybridization, slides were washed with Gene Expression Wash Buffer 1 and 2 (Gene Expression Wash Buffer Pack, Agilent).

scan_protocol: Scanned on Agilent G4900DA SureScan Microarray Scanner scanner (ScanRegion FullAgilentSlide, Scan resolution 3 μm, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: Agilent Feature Extraction Software (version 11.0.1.1) was used for image quantification, background subtraction, and LOWESS normalization.

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 μg RNA using the One-Color Low Input Quick-Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 .

growth_protocol: Endometrial organoids were propagated with the serum-free media Advanced DMEM/F-12 supplemented with 50 ng/ml EGF, 250 ng/ml R-spondin1, 100 ng/ml Noggin, 10 μM Y27632, 1 μM Jagged-1 and 2.5 μM CHIR-99021, L-glutamine solution, penicillin/streptomycin, and amphotericin B suspension.

extract_protocol: RNA was prepared using RNeasy Mini Kit  (QIAGNE, Venlo, Netherlands) following the manufacture's recommendations.

value: uterine corpus

tag: gender

value: female

tag: histology of subcutaneous tumor

value: pre-inoculation

tag: cell type


title: Em organoid_K5_#6_after

description:
Em organoid_K5_#6_after
Gene expression of subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss, #6

source_name: Subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss, #6

value: carcinosarcoma


title: Em organoid_KC_#4_before

description:
Em organoid_KC_#4_before
Gene expression of Em organoids with KrasG12D and shCdkn2a before subucutaneous inoculation, #4


title: Em organoid_K5_#6_before

description:
Em organoid_K5_#6_before
Gene expression of Em organoids with KrasG12D and Trp53 loss before subcutaneous inoculation, #6


title: Em organoid_K5_#5_after

description:
Em organoid_K5_#5_after
Gene expression of subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss, #5

source_name: Subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss, #5


title: fetal start rep2 [ATAC-seq]

description:
processed data file: FS.fragment.bw
15008X1_180420_D00550_0503_BCCFN4ANXX_S249_L007

data_processing:
Fastq files were aligned with Novocraft novoalign (v3.8.2) to the Ov3 genome allowing for one random multiple alignment. Standard Illumina sequence adapters were removed during alignment with Novoalign.
Samples were processed with MultiRepMacsChIPSeq pipeline (https://github.com/HuntsmanCancerInstitute/MultiRepMacsChIPSeq), version 12. Duplicate alignment pairs were subsampled to a final overall rate of 10%, alignments were filtered for mapping quality > 1, and pairs filtered against a repeat masker (UCSC) intervals and known high copy intervals.
Coverage tracks were generated by shifting the each alignment 5' coordinate by -10 bp and extending by 20 bp, resulting in 20 bp pileup centered over the 5' alignment position. Replicates were depth-normalized to 1 million reads and averaged together.
Genome_build: UCSC Ov3
Supplementary_files_format_and_content: BigWig normalized coverage tracks representing 20 bp pileup centered over the 5' alignment coordinate or tagmentation cut site.

source_name: right lower lung lobe_fetal start

extract_protocol:
Lung tissue removed, nuclei isolated from fresh lung tissue, tagmentation of 100K nuclei carried out at 37 C for 30 minutes with 10 ul Illumina Tagmentation enzyme
ATAC-seq libraries prepared with Illumina Nextera library kit following the manufacturer's protocol

value: right lower lung lobe


title: NRS rep4 [ATAC-seq]

description:
processed data file: NRS.fragment.bw
15090X1_180511_D00550_0506_BCCE8CANXX_3

source_name: right lower lung lobe_NRS

value: noninvasive respiratory support


title: fetal start rep1 [ATAC-seq]

description:
processed data file: FS.fragment.bw
14920X4_180308_D00294_0380_ACC9K4ANXX_2


title: NRS rep5 [ATAC-seq]

description:
processed data file: NRS.fragment.bw
15090X4_180511_D00550_0506_BCCE8CANXX_4


title: fetal end rep1 [ATAC-seq]

description:
processed data file: FE.fragment.bw
15964X1_190412_D00550_0559_ACDL9LANXX_S18_L002

source_name: right lower lung lobe_fetal end


title: fetal end rep2 [ATAC-seq]

description:
processed data file: FE.fragment.bw
15964X4_190412_D00550_0559_ACDL9LANXX_S21_L003


title: IMV rep1 [ATAC-seq]

description:
processed data file: IMV.fragment.bw
14920X1_180302_D00294_0378_ACC8EBANXX_8


title: IMV rep2 [ATAC-seq]

description:
processed data file: IMV.fragment.bw
14920X7_180308_D00294_0380_ACC9K4ANXX_3


title: IMV rep4 [ATAC-seq]

description:
processed data file: IMV.fragment.bw
14824X5_180118_D00550_0491_BCBTEEANXX_7


title: term rep3 [ATAC-seq]

description:
processed data file: Term.fragment.bw
15179X4_180615_D00550_0510_ACCJU1ANXX_8


title: NRS rep1 [ATAC-seq]

description:
processed data file: NRS.fragment.bw
14920X10_180308_D00294_0380_ACC9K4ANXX_4


title: IMV rep3 [ATAC-seq]

description:
processed data file: IMV.fragment.bw
14824X2_180118_D00550_0491_BCBTEEANXX_6


title: term rep2 [ATAC-seq]

description:
processed data file: Term.fragment.bw
15179X1_180615_D00550_0510_ACCJU1ANXX_7


title: FT organoid_K5_before

description:
FT organoid_K5_before
Gene expression of FT organoids with KrasG12D and Trp53 loss before subcutaneous inoculation

growth_protocol: Transduced FT organoids were propagated with the serum-free media Advanced DMEM/F-12 supplemented with 50 ng/ml EGF, 250 ng/ml R-spondin1, 100 ng/ml Noggin, 10 μM Y27632, 1 μM Jagged-1 and 2.5 μM CHIR-99021, L-glutamine solution, penicillin/streptomycin, and amphotericin B suspension.

extract_protocol: RNA was prepared using RNeasy Mini Kit  (QIAGEN, Venlo, Netherlands) following the manufacture's recommendations.

value: fallopian tube


title: FT organoid_A5_after

description:
FT organoid_A5_after
Gene expression of subcutaneous tumor-derived organoids with Apc580D and Trp53 loss

source_name: Subcutaneous tumor-derived organoids with Apc580D and Trp53 loss

value: cyst


title: FT organoid_K5_after

description:
FT organoid_K5_after
Gene expression of subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss

source_name: Subcutaneous tumor-derived organoids with KrasG12D and Trp53 loss


title: term rep1 [ATAC-seq]

description:
processed data file: Term.fragment.bw
15125X4_180615_D00550_0510_ACCJU1ANXX_6

source_name: right lower lung lobe_term


title: FT organoid_5_before

description:
FT organoid_5_before
Gene expression of FT organoids with Trp53 loss before subcutaneous inoculation


title: NRS rep3 [ATAC-seq]

description:
processed data file: NRS.fragment.bw
15008X4_180420_D00550_0503_BCCFN4ANXX_S252_L008


title: FT organoid_P5_before

description:
FT organoid_P5_before
Gene expression of FT organoids with Pik3caH1047R and Trp53 loss before subcutaneous inoculation


title: FT organoid_P5_after

description:
FT organoid_P5_after
Gene expression of subcutaneous tumor-derived organoids with Pik3caH1047R and Trp53 loss

source_name: Subcutaneous tumor-derived organoids with Pik3caH1047R and Trp53 loss

value: adenocarcinoma


title: FT organoid_A5_before

description:
FT organoid_A5_before
Gene expression of FT organoids with Apc580D and Trp53 loss before subcutaneous inoculation


title: miRNA set B from spot urine sample collected prior to surgery from patient B_AAN_U05

city: Lyon

first: Jiri

last: Zavadil

address: 150 cours Albert Thomas

country: France

institute: International Agency for Research on Cancer (IARC)

hyb_protocol: Quantitative TaqMan PCR amplification of miRNA-derived cDNAs was performed on the ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.

data_processing:
Cycle threshold (Ct) values were determined by automated threshold for each PCR reaction on the array using the ABI 7900HT qPCR system SDS v2.1 software
Ct values are provided, defined as the PCR cycle number at which the fluorescence of a PCR product was detected above the background signal by automated threshold for each reaction using the ABI 7900HT qPCR system SDS v2.1 software. No other adjustments have been done to the Ct readings in the Matrix data (linked on the Series record).

label: Fluorescein amidite (FAM)-labeled TaqMan probes

organism: Homo sapiens

source_name: Spot urine sample collected prior to surgery

label_protocol: MicroRNA (miRNA) expression levels were quantified using the TaqMan Human MicroRNA A Cards v2.1 + B Cards v3.0 (Applied Biosystems, Foster City, CA, USA) as per the manufacturer's instructions. cDNA was prepared from urinary cell-free RNA using the Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit and the Applied Biosystems Megaplex RT Human Pool A and RT Human Pool B primer mix. Pre-amplification was performed with Applied Biosystems TaqMan PreAmp Master Mix combined with the Applied Biosystems Megaplex PreAmp Primers, Human Pool A and Human Pool B. For PCR, the Applied Biosystems TaqMan Array Human MicroRNA A & B cards were used with Applied Biosystems TaqMan Universal Master Mix, No AmpErase UNG.

extract_protocol: 15 ml of each spot urine sample was centrifuged to sediment cells and larger debris, urine supernatant containing cell-free nucleic acids, including exosomic miRNAs was kept in long-term preservative (NorgenBiotek, Canada). Supernatant was subjected to additional high-speed centrifugation as to fractionate exosomic miRNAs from cellular debris and was spiked by 10 pM of control miRNA (derived form A. thaliana). The miRNA content from exosomes was isolated using Urine Exosome RNA Isolation Kit (NorgenBiotek, Canada).

tag: individual

value: patient B_AAN_U05

tag: age at surgery

value: 80 years

tag: sample type

value: urine


title: Em organoid_KL_before

description:
Em organoid_KL_before
Gene expression of Em organoids with KrasG12D and shLuc before subcutaneous inoculation


title: FT organoid_control_before

description:
FT organoid_control_before
Gene expression of Trp53flox/flox mice-derived FT organoids transduced with the control vector pLKO.1 before subcutaneous inoculation


title: NRS rep2 [ATAC-seq]

description:
processed data file: NRS.fragment.bw
14973X2_180406_D00550_0501_ACCFH6ANXX_2

tag: animal id


title: miRNA set B from spot urine sample collected 3 months after surgery from patient B_AAN_U03

scan_protocol: ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA)

source_name: Spot urine sample collected 3 months after surgery

value: patient B_AAN_U03

value: male

value: 76 years


title: miRNA set B from spot urine sample collected prior to surgery from patient B_AAN_U03


title: miRNA set B from spot urine sample collected prior to surgery from patient B_AAN_U04

value: patient B_AAN_U04


title: miRNA set B from spot urine sample collected 6 weeks after surgery from patient B_AAN_U03

source_name: Spot urine sample collectedat 6 weeks after surgery


title: Em organoid_control_before

description:
Em organoid_control_before
Gene expression of KrasG12D/+ and Trp53flox/flox mice-derived Em organoids before subcutaneous inoculation


title: miRNA set B from spot urine sample collected 6 weeks after surgery from patient B_AAN_U02

value: patient B_AAN_U02

value: 78 years


title: miRNA set B from spot urine sample collected prior to surgery from patient B_AAN_U02

value: female


title: miRNA set B from spot urine sample collected 3 months after surgery from patient B_AAN_U01

value: patient B_AAN_U01


title: Em organoid_KC_#4_after

description:
Em organoid_KC_#4_after
Gene expression of subcutaneous tumor-derived organoids with KrasG12D and shCdkn2a, #4

source_name: Subcutaneous tumor-derived organoids with KrasG12D and shCdkn2a, #4

value: uterine corpus

value: sarcoma and cyst


title: miRNA set A from spot urine sample collected prior to surgery from patient B_AAN_U03


title: miRNA set A from spot urine sample collected 6 weeks after surgery from patient B_AAN_U03


title: miRNA set A from spot urine sample collected prior to surgery from patient B_AAN_U04


title: miRNA set A from spot urine sample collected prior to surgery from patient B_AAN_U05


title: miRNA set A from spot urine sample collected 3 months after surgery from patient B_AAN_U03


title: miRNA set A from spot urine sample collected prior to surgery from patient B_AAN_U02


title: miRNA set A from spot urine sample collected prior to surgery from patient B_AAN_U01


title: miRNA set B from urothelial carcinoma, patient B_AAN_22

source_name: Surgically dissected urothelial carcinoma

label_protocol: MicroRNA (miRNA) expression levels were quantified using the TaqMan Human MicroRNA A Cards v2.1 + B Cards v3.0 (Applied Biosystems, Foster City, CA, USA) as per the manufacturer's instructions. cDNA was prepared from total RNA using the Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit and the Applied Biosystems Megaplex RT Human Pool A and RT Human Pool B primer mix. Pre-amplification was performed with Applied Biosystems TaqMan PreAmp Master Mix combined with the Applied Biosystems Megaplex PreAmp Primers, Human Pool A and Human Pool B. For PCR, the Applied Biosystems TaqMan Array Human MicroRNA A & B cards were used with Applied Biosystems TaqMan Universal Master Mix, No AmpErase UNG.

extract_protocol: Total RNA was isolated from fresh samples of tumor and adjacent normal tissue using using miRNAeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and stored in RNAlater. All samples were treated with DNAse (Qiagen, Valencia, CA, USA). RNA samples were stored at −80°C. The quantity and purity of RNA was measured using NanoDrop 2000c (Thermo Scientific, Wilmington, DE, USA). Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA) was used to determine the RNA integrity number which ranged between 8-10.

value: patient B_AAN_22

value: 59 years

value: UTUC tumor


title: miRNA set B from spot urine sample collected prior to surgery from patient B_AAN_U01


title: miRNA set B from urothelial carcinoma, patient B_AAN_27

value: patient B_AAN_27

value: 75 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_21

value: patient B_AAN_21

value: 83 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_20

value: patient B_AAN_20

value: 80 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_26

value: patient B_AAN_26


title: miRNA set B from urothelial carcinoma, patient B_AAN_18

value: patient B_AAN_18

value: 76 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_07

value: patient B_AAN_07

value: 73 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_25

value: patient B_AAN_25

value: male


title: miRNA set B from urothelial carcinoma, patient B_AAN_13

value: patient B_AAN_13

value: female

value: 71 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_04

value: patient B_AAN_04

value: 74 years

value: UTUC tumor


title: miRNA set B from urothelial carcinoma, patient B_AAN_06

value: patient B_AAN_06

value: 78 years


title: miRNA set B from urothelial carcinoma, patient B_AAN_03

value: patient B_AAN_03

value: 68 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_26

source_name: Normal urothelial tissue from tumor surgery

value: patient B_AAN_26

value: 80 years

value: normal urothelium


title: miRNA set B from normal urothelial tissue, patient B_AAN_25

value: 75 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_21

value: patient B_AAN_21

value: 83 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_22

value: patient B_AAN_22

value: male

value: 59 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_20

value: patient B_AAN_20

value: female


title: miRNA set B from normal urothelial tissue, patient B_AAN_18

value: patient B_AAN_18

value: 76 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_19

value: patient B_AAN_19

value: 66 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_15

value: patient B_AAN_15

value: 85 years


title: miRNA set A from spot urine sample collected 6 weeks after surgery from patient B_AAN_U02

value: patient B_AAN_U02

value: 78 years

value: urine


title: miRNA set B from normal urothelial tissue, patient B_AAN_06

value: patient B_AAN_06

value: normal urothelium


title: miRNA set B from normal urothelial tissue, patient B_AAN_14

value: patient B_AAN_14


title: miRNA set A from spot urine sample collected 3 months after surgery from patient B_AAN_U01

value: patient B_AAN_U01


title: miRNA set B from normal urothelial tissue, patient B_AAN_03

value: patient B_AAN_03

value: male

value: 68 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_25

value: patient B_AAN_25

value: 75 years

value: UTUC tumor


title: miRNA set A from urothelial carcinoma, patient B_AAN_26

value: patient B_AAN_26

value: female

value: 80 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_27

value: patient B_AAN_27


title: miRNA set A from urothelial carcinoma, patient B_AAN_18

value: patient B_AAN_18

value: 76 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_27


title: miRNA set A from urothelial carcinoma, patient B_AAN_22

value: patient B_AAN_22

value: 59 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_13

value: patient B_AAN_13

value: 71 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_20

value: patient B_AAN_20


title: miRNA set A from urothelial carcinoma, patient B_AAN_07

value: patient B_AAN_07

value: 73 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_06

value: patient B_AAN_06

value: 78 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_04

value: patient B_AAN_04

value: 74 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_13

value: normal urothelium


title: miRNA set A from urothelial carcinoma, patient B_AAN_03

value: patient B_AAN_03

value: male

value: 68 years

value: UTUC tumor


title: miRNA set A from normal urothelial tissue, patient B_AAN_25

value: patient B_AAN_25

value: 75 years


title: miRNA set B from normal urothelial tissue, patient B_AAN_04

value: female


title: miRNA set A from normal urothelial tissue, patient B_AAN_20

value: 80 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_22

value: patient B_AAN_22

value: 59 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_27

value: patient B_AAN_27


title: miRNA set A from normal urothelial tissue, patient B_AAN_21

value: patient B_AAN_21

value: 83 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_15

value: patient B_AAN_15

value: 85 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_18

value: patient B_AAN_18

value: 76 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_06

value: patient B_AAN_06

value: 78 years

value: normal urothelium


title: miRNA set A from normal urothelial tissue, patient B_AAN_19

value: patient B_AAN_19

value: male

value: 66 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_13

value: patient B_AAN_13

value: female

value: 71 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_03

value: patient B_AAN_03

value: 68 years


title: miRNA set A from urothelial carcinoma, patient B_AAN_21

value: patient B_AAN_21

value: UTUC tumor


title: miRNA set A from normal urothelial tissue, patient B_AAN_26

value: patient B_AAN_26

value: 80 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_04

value: patient B_AAN_04

value: 74 years


title: miRNA set A from normal urothelial tissue, patient B_AAN_14

value: patient B_AAN_14

value: 76 years


title: ATAC-seq_TAM_2

city: Shanghai

first: Xuelong

address: No. 100 Banxia Road

country: China

institute: Chinese Academy of Sciences

description: TAMs isolated from human PDAC tissues

data_processing:
ChIP-Seq and ATAC-seq data sets were trimmed and aligned to hg19 genome using Bowtie2. Reads from RNA-seq experiments were aligned to hg19 genome using STAR.
Duplicate reads were removed with SAMtools.
MACS2 was used to identify regions of ChIP-seq and ATAC-seq peaks; The mRNA expression of a gene was quantified by FPKM based on RefSeq gene annotation using Cuffdiff and R packages.
The top 10000 peaks were used for known motif enrichment analysis, which were performed using HOMER (v4.9).
Genome_build: hg19
Supplementary_files_format_and_content: The bw (BigWig) files were generated using deepTools.

source_name: TAMs cells

extract_protocol: (1) The RNA-Seq assay was performed as previously described. Total RNA product was purified using RNeasy Mini Kit (QIAGEN, Germany) and run-on Agilent Bioanalyzer 4200 to assess RNA integrity. The total RNA‐seq library preparation from 1 μg of total RNA was performed with TruSeq RNA Sample Prep Kit (Illumina, USA) according to the manufacturer's instructions. (2) The ChIP assay was performed as previously described. H3K27ac (ab4729, Abcam, USA), H3K27me3(9733S, CST, USA), CTCF (Millipore, 07-729), H3K9ac (9649, CST, USA), H3K4me (5326, CST, USA), EP300 (ab275378, Abcam, USA) were used for ChIP experiments. For each sample, 100 million crosslinked cells were lysed to prepare nuclear extracts. After chromatin shearing by sonication for 10 min, lysates were incubated on sky wheel overnight at 4°C with protein A Dynabeads (Invitrogen, USA) coupled with 3-6 μg of antibodies. After immunoprecipitation, beads were washed and recovered using magnets. DNA was reverse- cross‐linked at 55°C for 4 hours. After 4 hours, DNA product was purified with QIAquick PCR Purification Kit (QIAGEN, USA). DNA was quantitated using the Qubit dsDNA HS assay and a Qubit 2.0 Fluorimeter (Invitrogen). For ChIP-seq, 5 ng-50ng of purified DNA product were used to generate the DNA library using a VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme Biotech, China) and the Illumina HiSeq X Ten was used to sequence the paired-end module and with 150 bp reads on each end (Novogene Biotech, China). For ChIP-qPCR, ChIP products which were used to RT-PCR were performed to amplify the pcr products for 40 cycles using the following PCR conditions: 95 °C for 3 min; and thermocycling at 95°C for 15 s, 60 °C for 60 s, following by 4 °C forever. (3) The ATAC-seq assay was performed as previously described. 1 million fresh cells (THP-1 co-cultured with PANC-1 treated with PMA, TAMs and monocytes) were lysed in 100μL lysis buffer per sample (10 mM Tris-HCl at pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) for 10 min on ice to prepare the nuclei briefly. Immediately After 10 min, nuclei were spun for 5 min at 500 x g to remove the supernatant. Then nuclei were incubated at 37 °C with the tagmentation buffer and Tn5 transposome (Vazyme Biotech, China) for 30 min. After the 30 min, ended buffer was added directly into the reaction to quit the tagmentation. Products which were inputted to PCR were performed to amplify the library for 11 cycles using the following PCR conditions: 72 °C for 3 min; 98 °C for 30 s; and thermocycling at 98 °C for 15 s, 60 °C for 30 s and 72 °C for 3 min; following by 72 °C 5 min. After the PCR reaction, DNA libraries were purified with DNA Clean Beads (Vazyme Biotech, China) to remove invalid primer dimmers and quantitate used Qubit 2.0 Fluorimeter (Invitrogen, USA) to quantitate. (4) All eligible DNA libraries were sequenced on the Illumina HiSeq X Ten with 150 bp paired-end reads.

value: untreated


title: ATAC-seq_shNC

description: THP1 co-cultured with PANC1

source_name: THP1+PANC1 cell lines

tag: cell line

value: shNC


title: ATAC-seq_MONO_1

description: Mononuclear cells isolated from peripheral blood

source_name: MONO cells

tag: treatment


title: ChIP-seq_CTCF_shNC

description: THP1 co-cultured with PANC1

tag: antibody

value: CTCF


title: ATAC-seq_TAM_1

description: TAMs isolated from human PDAC tissues


title: ATAC-seq_MONO_2


title: ChIP-seq_Input

description: THP1 co-cultured with PANC1


title: ChIP-seq_H3K9ac_shNC

description: THP1 co-cultured with PANC1


title: ChIP-seq_H3K9ac_shCTCF

description: THP1 co-cultured with PANC1

value: shCTCF


title: ChIP-seq_H3K27ac_shCTCF

description: THP1 co-cultured with PANC1


title: ChIP-seq_H3K27ac_shNC

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_H - Control

city: Toulouse

first: Philippe

last: Pinton

address: 180 Chemin de Tournefeuille

department: TOXALIM

institute: INRAE

hyb_protocol: 600 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 10x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Porcinet 60K_DEC2011 GE microarray (8X60K, Design 037880  enclosed in Agilent SureHyb-enabled hybridization chambers for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed sequentially in Wash buffer 1 (Agilent Technologies, 1 min) and Wash buffer 2 (Agilent Technologies, 37°C, 1 min).

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

data_processing: The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent Technologies, Santa Clara, CA) using default parameters (protocol GE1_1010_Sep10 and Grid: 037880_D_F_20120213). All subsequent data analyses were done under R (www.r-project.org) using packages of Bioconductor (www.bioconductor.org). Raw data (median of pixels intensity) were imported into R using the read.maimages function from the limma package with the following weight function (assigning a weight of 1 or 0 to each spot): myfunw<-function(x) {okType<-x$ControlType==0; okFoundGreen<-x$gIsFound==1; okPos=x$gIsPosAndSignif==1; okWellAbove<- x$gIsWellAboveBG==1; as.numeric(okType & okFoundGreen & okPos & okWellAbove);} We selected the spots with a minimal weight of 1 for 13 out of 16 microarrays or with a minimal weight of 4 per group from at least one experimental group. At this step, 41437 spots out of 61625 were selected. Data were then stored in an ExpressionSet object and normalized by the quantile method using the normalize.quantiles function from the preprocessCore R library. Replicated probes on the array (identical ProbeName) were resolved by taking the median normalized signal of each set of replicated probes. The resulting matrix has 41436 rows each corresponding to a unique ProbeName (provided as data Matrix).

organism: Sus scrofa

source_name: Intestinal explants

label_protocol: For each sample, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by Agencourt RNAClean XP (Agencourt Bioscience Corporation, Beverly, Massachusetts) purification. Dye incorporation and cRNA yield were checked using Dropsense™ 96 UV/VIS droplet reader (Trinean, Belgium).

growth_protocol: Explants obtained from biopsy punches (6 mm diameter) were cultured in 6-wells plates in Williams E medium supplemented with ITS, antibiotics and alanine glutamine at 39°C during 4 h.

extract_protocol: RNA were extracted with phenol chloroform technique, quantified with a Nanodrop device and their quality was assessed with a Tapestation 4200 device. RIN values were between 6.4 and 8.1.

treatment_protocol: Explants were cultured in control medium contaning vehicle (DMSO) or deoxynivalenol (10 µM)

tag: animal

value: H

value: Control


title: ATAC-seq_shCTCF

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_F - DON

value: F

value: deoxynivalenol-contaminated cell culture medium


title: ChIP-seq_H3K27me3_shNC

description: THP1 co-cultured with PANC1


title: ChIP-seq_H3K27me3_shCTCF

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_H - DON


title: Intestinal explants - Animal_B - Control

value: B


title: Intestinal explants - Animal_C - DON

value: C


title: Intestinal explants - Animal_E - DON

value: E


title: Intestinal explants - Animal_B - DON


title: Intestinal explants - Animal_G - DON

value: G


title: RNA-seq_shCTCF_2

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_A - Control

value: A


title: Intestinal explants - Animal_G - Control


title: C4-gan

city: Nanjing

first: Yong

last: Su

address: Weigang 1#

institute: Nanjing Agricultural University

description: Liver tissue treated with physiological saline

data_processing:
Using the Illumina paired-end RNA-seq approach
The clean reads were mapped to the reference pig genome (Sus scrofa 10.2) by the mapping tool TopHat (version: 2.0.9)
StringTie and Ballgown was used to estimate the expression levels of all transcripts. StringTie was used to perform expression level for mRNAs by calculating FPKM
The differentially expressed mRNAs and genes were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package – Ballgown.
Genome_build: ftp://ftp.ensembl.org/pub/release-83/fasta/sus_scrofa/dna/Sus_scrofa.Sscrofa1 0.2.dna.toplevel.fa.gz
Supplementary_files_format_and_content: xls include FPKM values for each Sample

source_name: Liver tissue

growth_protocol: This study was approved by the Nanjing Agricultural University Animal Care and Use Committee (Nanjing, Jiangsu province, China). All animal procedures were previously described by Chen et al. (15). In detail, 12 growing barrows (Duroc × Landrace × Large White) from the research pig farm  were used. Each barrow was placed in an individual pen and received a commercial diet (metabolizable energy = 14.20 MJ/kg; crude protein = 16.8%) and water ad libitum. A medical polyethylene cannula (Braintree Scientific Inc, Braintree, MA) was surgically installed into the internal jugular vein to enable the infusion of SB.

extract_protocol:
Total RNA was extracted using RNAiso Plus Total RNA extraction reagent (Cat#9109, TAKARA) following the manufacturer’s instructions
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Twelve crossbred barrows (Duroc × Landrace × Large White, 60 days of age) were fitted with a medical polyethylene cannula via internal jugular vein and were daily infused with 10 ml SB (200 mmol/l) or the same volume of physiological saline.The infusion from each pig was performed in 5 mins. After a 7-day experimental period, all pigs were weighed and euthanized after 12 h of fasting. The 10th-rib longissimus dorsi muscle and dorsal subcutaneous adipose tissue were collected and preserved in liquid nitrogen for further transcriptomic analysis.


title: Intestinal explants - Animal_C - Control


title: Intestinal explants - Animal_F - Control


title: Intestinal explants - Animal_D - Control

value: D


title: Intestinal explants - Animal_E - Control


title: RNA-seq_shCTCF_1

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_A - DON


title: RNA-seq_shNC_1

description: THP1 co-cultured with PANC1


title: Intestinal explants - Animal_D - DON


title: C3-ji

description: Muscle tissue treated with physiological saline

source_name: Muscle tissue


title: C3-gan

value: Liver tissue treated with physiological saline


title: C3-zhi

description: Fat tissue treated with physiological saline

source_name: Fat tissue


title: C2-ji

value: Muscle tissue treated with physiological saline


title: C2-gan


title: C2-zhi

value: Fat tissue treated with physiological saline


title: C4-zhi


title: B4-ji

description: Muscle tissue treated with sodium butyrate


title: C4-ji


title: B2-ji

value: Muscle tissue treated with sodium butyrate


title: RNA-seq_shNC_2

description: THP1 co-cultured with PANC1

value: shNC


title: B4-gan

description: Liver tissue treated with sodium butyrate


title: B1-zhi

description: Fat tissue treated with sodium butyrate

value: Fat tissue treated with sodium butyrate


title: B1-gan

value: Liver tissue treated with sodium butyrate


title: B2-gan


title: B1-ji


title: B4-zhi

description: Fat tissue treated with sodium butyrate


title: Pro08

city: Utrecht

first: Michael

last: VanInsberghe

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

description:
processed data files: EE_metadata.csv.gz
processed data files: EE_RPFcounts.csv.gz
processed data files: EE_Pro_layout.csv.gz

data_processing:
See github repository https://github.com/mvanins/scRiboSeq_manuscript for code and detailed instructions.
Basecalls were performed on the Illumina NextSeq 500. Reads from each plate were demultiplexed using bcl2fastq (version 2.20.0.422) with --use-bases-mask Y*,I*,Y* --no-lane-splitting --mask-short-adapter-reads 0 --minimum-trimmed-read-length 0. Read 1 contains the insert and Read 2 contains the cell barcode.
Next, the UMI was extracted from the first 10 bases of read 1 and concatenated to the start of the cell barcode. Adapter sequences were then trimmed from read 1 using cutadapt (version 2.7) with -m 15: -a TGGAATTCTCGGGT. Trimmed reads were aligned to the reference genome using STARSolo (version 2.7.3a_2020-05-22) with a 50-base overhang (--sjdbOverhang 50) with the following parameters: --seedSearchStartLmax 10 –alignIntronMax 1000000 --outFilterScoreMin 0 --outFilterMultimapNmax 1 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outFilterMismatchNmax 5. Aligned reads were deduplicated with UMI-tools (version 1.0.1) using --spliced-is-unique --per-cell --read-length --no-sort-output, and sorted using sambamba (version 0.7.1). Finally, aligned and deduplicated reads were parsed to extract the mapping coordinates and the reference sequence contexts around both the 5’ and 3’ ends of the reads.
Count tables report the number of unique RPFs aligning within any annotated coding sequence for each gene. Reads were only counted if they aligned to the correct strand, if the trimmed length was between 30 and 45 nt long, and if the trimmed length was equal to the mapped length. Additionally, for these counts, the coding sequence was expanded to include 25 nt upstream and downstream of the start and stop codons, respectively.
A random forest model was trained to predict the A-site location within an RPF read based on the footprint length and the sequence context around the 5’ and 3’ ends. The model was implemented in R (version 3.6.3) using ranger (version 0.12.1) with mlr (version 2.17.1) wrappers for training, tuning, assessment, and prediction. The model was trained on reads spanning a stop codon that satisfied the counting requirements listed above. The number of nucleotides between the 5’ end of these reads and the annotated stop codon was used for training.
hTERT-RPE1-FUCCI cells were only included in downstream analyses if they had more than 4500 unique reads aligning to protein-coding genes, and if more than 85% aligned to coding sequences.
Supplementary_files_format_and_content: Metadata file: Cell-level metadata for cells passing QC. Column Headers: CB: Cell barcode. treatment: Culture/starvation condition prior to sequencing. well: Well location in plate. plate: Plate name. cds: Number of unique reads aligning to coding sequences. utr5: Number of unique reads aligning to 5' UTRs. utr3: Number of unique reads aligning to 3' UTRs. cell_total: Total number of unique reads aligning to protein-coding genes. cds_frac: Fraction of unique total reads aligning to CDS.
Supplementary_files_format_and_content: Count file: Cell-level count tables denoting the number of unique reads aligned to CDS regions for each gene.
Supplementary_files_format_and_content: Plate layout file: Description of the plate layout. Column Headers: well: well location in plate. CB: cell barcode for this well. fraction: Cell fraction/population/treatment.
Genome_build: GRCm38.p6

source_name: Mouse enteroendocrine cells from the proximal section of the intestine.

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: CGGAAT; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/

treatment_protocol: Mouse enteroendocrine cells were isolated from the intestines of Neurog3Chrono mice, closely following the methods outlined by Gehart et al. Cell 2019 doi:10.1016/j.cell.2018.12.029. Briefly, mouse small intestines were harvested, cleaned, flushed with PBS0, and separated into proximal, medial, and distal sections. Pieces were cut open and villi were scraped off with a glass cover slip and discarded. Tissue pieces were then washed in cold PBS0 before transferring to PBS0 with 2 mM EDTA (Gibco), incubated at 4 °C for 30 minutes on a roller, and then vigorously shaken. Detached crypts were pelleted, resuspended in warm TrypLE Select (Gibco), and mechanically disrupted by pipetting to generate single-cell suspensions. Single-cell suspensions were washed 2× in Advanced DMEM/F12 (Gibco), strained with a 20-µm mesh, and resuspended in Advanced DMEM/F12 containing 4 mM EDTA and 1 µg/mL DAPI for sorting. In sorting, doublets, debris, and dead cells were excluded by gating forward and side scatter in combination with the DAPI channel. The measurements of dTomato and mNeonGreen were used to select enteroendocrine cells expressing the Neurog3Chrono reporter and DAPI was used to exclude dead cells.

value: enteroendocrine

tag: strain

tag: tissue lineage

value: Enteroendocrine, intestinal epithelium

tag: genetic tag

tag: cell fraction

value: Proximal intestine


title: Dis01

description:
processed data files: EE_metadata.csv.gz
processed data files: EE_RPFcounts.csv.gz
processed data files: EE_Dis_layout.csv.gz

source_name: Mouse enteroendocrine cells from the distal section of the intestine.

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: TCATTC; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/

value: Distal intestine


title: Mid02

description:
processed data files: EE_metadata.csv.gz
processed data files: EE_RPFcounts.csv.gz
processed data files: EE_Mid_layout.csv.gz

source_name: Mouse enteroendocrine cells from the medial section of the intestine.

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: ACTTGA; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/

value: Medial intestine


title: Pro02

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: GCCAAT; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: Pro05

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: CGATGT; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/