title: SR205

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.
adapters: barcode: TGACCA; 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: CT26.indisulam_1uM.B

status: Public on Jun 30 2021

type: SRA

city: Seattle

first: James

middle: Douglas

last: Thomas

state: WA

address: 1100 Fairview Ave. N., B3-111

department: Computational Biology

country: USA

institute: Fred Hutchinson Cancer Research Center

description: RNA-seq of cell line treated with the indicated compound

library_source: transcriptomic

data_processing:
Illumina Casava software was used for base calling.
For RNA-seq, reads were aligned to the mouse transcriptome using RSEM followed by mapping to the mouse genome (mm10) using Bowtie and TopHat.
RNA splicing was quantified using MISO.
Genome_build: mm10 for mouse data
Supplementary_files_format_and_content: The final processed data is found in the file "d.psi.txt"

molecule: polyA RNA

organism: Mus musculus

extract_protocol:
RNA was extracted from cells of interest using an RNeasy kit (Qiagen, Frederick, MD) and quantified using a NanoDrop 8000 (ThermoFisher Scientific); a minimum of 500 ng of high quality RNA (as determined by Agilent Bioanalyzer) per sample or replicate was used for library preparation.
Poly(A)-selected, stranded Illumina libraries were prepared with a modified TruSeq protocol and sequenced on the Illumina HiSeq 2000

treatment_protocol: Cell lines were treated with splicing inhibitors at the indicated concentrations for 96 hours in vitro, harvested and washed three times with PBS in excess to remove all drug, and then used for downstream analyses, continued growth in vitro or tumor challenge in vivo into syngeneic animals.

tag: cell line


title: B73_lowN_Rep1

city: New York

first: Chia-Yi

last: Cheng

state: NY

address: 12 Waverly Place

department: Biology

institute: New York University

description: processed data file: NutriNet.maize-leaf.featureCounts.txt

data_processing:
Base calling: (Arabidopsis) Picard Tools version 2.17.11 MINIMUM_QUALITY=2 INCLUDE_NON_PF_READS=false APPLY_EAMSS_FILTER=false. (Maize) bcl2fastq v2.17.1.14 Conversion Software (Illumina).
Removing optical duplicates: Clumpify version 37.24 (Arabidopsis) clumpify.sh -Xmx48g in=in.fq.gz out=out.fq.gz overwrite=t dedupe reorder addcount optical dupedist=40. (Maize) clumpify.sh -Xmx60g in=in.fq.gz out=out.fq.gz overwrite=t dedupe reorder addcount optical dupedist=2500.
Removing adapters: BBDuk version 37.24 (Arabidopsis). (Maize) bbduk.sh in=in.fq.gz out=out.fq.gz overwrite=t ref=~/package/bbmap/resources/adapters.fa ordered=t ktrim=r k=25 mink=11 minlength=35.
Aligning to the genome: BBMap version 37.24 (Arabidopsis). (Maize) bbmap.sh -Xmx56g in=input.fq.gz ordered=t overwrite=t outm=out.sam
Assigning reads: subread v1.5.1 (Arabidopsis) featureCounts -T 1 -s 2 -O -a Araport11.gtf -o out.featureCounts input.sam (Maize) featureCounts -T 1 -R -s 0 -O -a Zea_mays.AGPv4.32.gtf -o out.featureCounts input.sam
Genome_build: (Arabidopsis) TAIR10, (Maize) Zm-B73-REFERENCE-GRAMENE-4.0
Supplementary_files_format_and_content: *.featureCounts.txt: Tab-delimited text files include raw read counts.

molecule: total RNA

organism: Zea mays

source_name: Leaf

extract_protocol:
(Arabidopsis) Total RNA was extracted from leaf samples using Direct-zol MiniPrep by Zymo Research.
(Arabidopsis) NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina.
(Maize) RNAs were isolated with a CTAB, acidified phenol-chloroform extraction, and isopropanol precipitation.
(Maize) TruSeq Stranded mRNAseq Sample Prep kit.

tag: tissue

value: Leaf tissue taken two inches from the base of leaf 13, which is the leaf subtending the top ear

tag: age

value: R1 stage

tag: genotype

tag: treatment

value: Low nitrogen


title: B73_lowN_Rep2


title: B73_highN_Rep3

value: High nitrogen


title: CT26.dmso.H


title: CT26.dmso.I


title: CT26.indisulam_1uM.A


title: B_uninfected

city: Baltimore

first: Monika

last: Looney

state: MD

address: 1503 E Jefferson Street

institute: Johns Hopkins University

data_processing:
read alighment using HISAT2
sorting and SAM/BAM conversion using samtools
read assembly, quantification, merging using Stringtie
transcript abundance estimation using Ballgown
differential expression analysis using prepDE.py and DESeq2
Genome_build: GRCh38_latest_genomic.fna
Supplementary_files_format_and_content: CSV files containing gene or transcript count matrix

organism: Homo sapiens

source_name: Primary monocyte-derived macrophages

growth_protocol: Monocytes were extracted from platelet-depleted blood from healthy human donors by Ficoll density gradient centrifugation and plastic adherence. Monocytes were allowed to adhere for 4 hours, after which non-adherent non-monocytes were washed away in 1X phosphate-buffered saline. Monocytes were differentiated for one week. Cells were cultured in RPMI-1640 + 10% fetal bovine serum + 4mM L-glutamine.

extract_protocol:
Total RNA was isolated using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Cat # 80204) following manufacutrer instructions.
RNA sequencing libraries were prepared using the Illumina TruSeq RNA library prep kit v2 (Illumina, Cat # RS-122-2011)

treatment_protocol: After 1 week differentiation, macrophages were either left uninfected or infected with Mtb strain H37Rv at a MOI of 10 for 48 hours.

tag: cell type

value: Primary monocyte-derived macrophages

tag: donor

value: B

tag: moi

value: uninfected


title: B73_highN_Rep2


title: SR201

city: Utrecht

first: Michael

last: VanInsberghe

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

description:
processed_data_file: RPE-FUCCI_metadata.csv.gz
processed_data_file: RPE-FUCCI_RPFcounts.csv.gz
processed_data_file: RPE_SR2_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: hg38

source_name: Interphase hTERT RPE1 FUCCI cells

growth_protocol: RPE1 hTERT FUCCI cells (doi: 10.1073/pnas.1322021111) were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1× GlutaMAX (Gibco) and 1× Pen-Strep (Gibco) at 37 °C 5% CO2.

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.
adapters: barcode: TTAGGC; 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: For the interphase fraction, 7.5E4 cells were plated in a MW-6 and collected by trypsinization (TrypLE, Gibco) 36 hours later. DAPI (ThermoFisher Scientific) was added to cultures as a viability marker, and only viable cells were sorted.

value: hTERT RPE-1

tag: tissue lineage

value: Retina, eye; pigmented epithelium

tag: genetic tag

value: Fluorescent Ubiquitination-based Cell-Cycle Indicators (FUCCI)

tag: cell fraction

value: Interphase cells

value: Interphase treatment


title: SR202

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.
adapters: 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: B73_highN_Rep1


title: CT26.ms023_1uM.G


title: KO03

description:
processed_data_file: RPE-FUCCI_metadata.csv.gz
processed_data_file: RPE-FUCCI_RPFcounts.csv.gz
processed_data_file: RPE_KO_layout.csv.gz

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.
adapters: barcode: TCCCGA; 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: TaH-LPG

city: Edinburgh

first: Stephen

last: Larcombe

state: Lothian

address: Charlotte Auerbach Road

country: United Kingdom

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 taurus

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: holstein fresian breed

tag: passage

value: harvested passage 6-8


title: KO04

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.
adapters: barcode: CAGATC; 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: CT26.dmso.G


title: CT26.dmso.C


title: KO02

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.
adapters: barcode: TATAAT; 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: CT26.dmso.B


title: KO01

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.
adapters: barcode: CTAGCT; 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: TaH130


title: SR204

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.
adapters: 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/


title: CT26.dmso.A


title: SR203

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.
adapters: barcode: CTTGTA; 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: TaH12852


title: TaH12810


title: TaH12886


title: B16-F10.ms023_1uM.H


title: M-H2Bub1

city: Wuhan

first: Lai

last: fengling

address: Wuhan University, Wuchang District, Hubei Province

country: China

institute: Wuhan university

library_source: genomic

data_processing:
Data filtering includes removing adaptor sequences, contamination and low-quality reads from raw reads.
Clean Parameter: SOAPnuke filter -l 15 -q 0.2 -n 0.01 -Q 2 -c 21
Clean data was then mapped to the reference genome by SOAPaligner/SOAP2 (Version: 2.21t)
MACS (Model-based Analysis for ChIP -Seq, version: MACS-1.4.2) : The candidate Peak region was extended to be long enough for modeling. Dynamic Possion Distribution was used to calculated p-value of the specific region based on the unique mapped reads. The region would be defined as a Peak when p-value < le-05.
Genome_build: assembly M_albus_1.0
Supplementary_files_format_and_content: peak text files

molecule: genomic DNA

organism: Monopterus albus

source_name: testis

growth_protocol: adult tissue

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
DNA-end repair, 3'-dA overhang and ligation of methylated sequencing adaptor.PCR amplification and size selection.Qualified library for sequencing.

treatment_protocol: untreatment

value: testis

value: untreatment

tag: antibody

value: Rabbit antibody to Ubiquityl-Histone H2b (Lys 120) (Cat# 5546s)


title: B16-F10.ms023_1uM.G


title: Mit08

description:
processed_data_file: RPE-FUCCI_metadata.csv.gz
processed_data_file: RPE-FUCCI_RPFcounts.csv.gz
processed_data_file: RPE_Mit_layout.csv.gz

source_name: hTERT RPE1 FUCCI cells from either interphase or mitotic shake-off fractions.

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.
adapters: barcode: GAGTGG; 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: For the interphase fraction, 7.5E4 cells were plated in a MW-6 and collected by trypsinization (TrypLE, Gibco) 36 hours later. For the mitotic fraction, 3E6 cells were plated in a 145 mm dish and were harvested 36 hours later by gently tapping the culture dish and collecting the media (otherwise known as a mitotic shake-off). DAPI (ThermoFisher Scientific) was added to cultures as a viability marker, and only viable cells were sorted.

value: Mitotic shake-off and interphase cells. Specific cell fraction for each well is defined in RPE_Mit_layout.csv.gz

value: Interphase and Mitotic treatments


title: B16-F10.indisulam_1uM.C


title: PSD3: Planktonic-shedding_Rep3

city: Khon Kaen

first: Supaksorn

last: Chattagul

state: Khon Kaen

address: 123 Moo 16 Mittraphap Rd., Nai-Muang

department: Biochemistry

country: Thailand

institute: Khon Kaen University

data_processing:
The RNA-Seq libraries were sequenced using a single-end reads mode with an Illumina HiSeq 1500 platform (Illumina, USA). The raw RNA-Seq data were formatted in the FASTQ file. The FASTQ datasets were subjected to evaluate the quality control with FastQC [v0.11.4], and then the reads were trimmed by using Trimmomatic [v0.35] with a leading and trailing minimum score of 3 and a 4-base sliding window minimum score of 15. Post-trimmed reads were aligned to a reference of the chromosome I and II of the B. pseudomallei strain K96243 (GenBank accession no. NC006350.1 and NC006351.1) (NCBI, NIH) using Bowtie2 [v2.1.0]. The differential of gene-levels count was determined with Cuffdiff, a part of the Cufﬂinks package [v2.1.1], and a total of the normalized-genes output was reported as fragments per kilobase per million mapped reads (FPKM) with q-value of < 0.05 and 2-fold change above 1 (Log2 scale).
Genome_build: NC006350.1 and NC006351.1
Supplementary_files_format_and_content: Cuffdiff files include FPKM values for each Sample ...
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Burkholderia pseudomallei K96243

source_name: Planktonic-shedding cell

growth_protocol: Overnight bacterial culture was grown in the enrichment liquid Luria-Bertani (LB) broth at 37°C with shaking until the exponential phase was reached. Planktonic-free cell pellets were recovered by centrifugation at 8,000×g for 5 min. Bacterial biofilm cells were cultured in the modified Vogel and Bonner’s medium (MVBM), in 24-well plates (Thermo Fisher Scientific, USA). An initial inoculum was diluted in MVBM to obtain 1.0×107 CFU mL-1 and grown at 37°C with shaking at 100 rpm for 24 hr. Bacterial biofilm was rinsed with phosphate buffered saline (PBS, pH 7.4) for 3 times and bacterial cells were collected by scrapping in TRIzol reagent. Planktonic shedding cells were obtained from MVBM culture using the modification of Nunc-Immuno TSP with 96 pins at the lid (Thermo Fisher Scientific, USA), Bacterial biofilm that formed was rinsed with PBS for 3 times and transferred the lids to 96-wells plate that contained enrich medium and incubated at 37°C for another 24 hr. The cell pellets in the medium were processed the same as planktonic cell and cultured in LB for 24 hr.

extract_protocol:
Total RNA was extracted from bacterial pellets and performed using a TRIzol reagent with the Purelink RNA mini kit (Ambion, USA). RNA integrity was evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The total RNA sample 2.5 μg was used to construct the RNA libraries. Total RNA in each sample, after removal of the 23S and 16S ribosomal RNA (rRNAs) with Ribo-Zero Gram-negative Bacteria rRNA removal kit (Epicentre Technologies, USA), was purified by an ethanol precipitation. The cDNA libraries were assembled from the fragmented purified rRNA-depleted RNA using a ScriptSeq v2 RNA-Seq Library Preparation kit (Epicentre Technologies, USA) and purified using a QIAquick PCR purification kit (QIAGEN, USA). An Illumina Indexed-adapter sequence was ligated to di-tagged cDNA to generate the amplified libraries as per the RNA sample preparation protocol (Illumina, USA). Excess nucleotides and PCR primers were eliminated from the libraries with a QIAquick PCR purification kit and the quantity and quality were examined using an Agilent 2100 Bioanalyzer.
cDNA libraries were prepared and constructed using a ScriptSeq v2 RNA-Seq Library Preparation kit (Epicentre Technologies, USA)

tag: strain

tag: growth condition

value: 24-hr Planktonic-shedding cell growing in Luria-Bertani broth (LB)


title: Mit04


title: Mit03

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.
adapters: barcode: ATCACG; 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: F-H2Bub1

source_name: ovary

value: ovary


title: Mit06

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.
adapters: barcode: GGCTAC; 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: Mit02

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.
adapters: barcode: TCGAAG; 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: PSD2: Planktonic-shedding_Rep2


title: B16-F10.indisulam_1uM.B

extract_protocol:
RNA was extracted from cells of interest using an RNeasy kit (Qiagen, Frederick, MD) and quantified using a NanoDrop 8000 (ThermoFisher Scientific); a minimum of 500 ng of high quality RNA (as determined by Agilent Bioanalyzer) per sample or replicate was used for library preparation.
Poly(A)-selected, stranded Illumina libraries were prepared with a modified TruSeq protocol and sequenced on the Illumina HiSeq 2000


title: B16-F10.indisulam_1uM.A


title: B3: Biofilm_Rep3

source_name: Biofilm cell

value: 24-hr biofilm growing in the modified Vogel and Bonner’s medium  (MVBM)


title: G012

description:
processed_data_file: RPE-FUCCI_metadata.csv.gz
processed_data_file: RPE-FUCCI_RPFcounts.csv.gz
processed_data_file: RPE_G0_layout.csv.gz

source_name: hTERT RPE1 FUCCI cells from either interphase or contact-inhibition G0 fractions.

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.
adapters: barcode: GTCCGC; 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: For the interphase fraction, 7.5E4 cells were plated in a MW-6 and collected by trypsinization (TrypLE, Gibco) 36 hours later. For the G0-arrested fraction, 1E5 cells were plated in a MW-24 and collected 72 hours later by trypsinization. DAPI (ThermoFisher Scientific) was added to cultures as a viability marker, and only viable cells were sorted.

value: Contact-inhibition G0 and interphase cells. Specific cell fraction for each well is defined in RPE_G0_layout.csv.gz.

value: Interphase and G0 treatments


title: Mit01

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.
adapters: barcode: CTCAGA; 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: hTERT RPE-1


title: Mit07

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.
adapters: barcode: AGTCAA; 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: Retina, eye; pigmented epithelium


title: B73_lowN_Rep3

extract_protocol:
(Arabidopsis) Total RNA was extracted from leaf samples using Direct-zol MiniPrep by Zymo Research.
(Arabidopsis) NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina.
(Maize) RNAs were isolated with a CTAB, acidified phenol-chloroform extraction, and isopropanol precipitation.
(Maize) TruSeq Stranded mRNAseq Sample Prep kit.

value: Leaf tissue taken two inches from the base of leaf 13, which is the leaf subtending the top ear

value: R1 stage

value: Low nitrogen


title: B16-F10.dmso.G


title: B16-F10.dmso.H


title: B16-F10.dmso.C


title: B16-F10.dmso.B


title: B1: Biofilm_Rep1


title: B2: Biofilm_Rep2


title: B16-F10.dmso.I