title: scRNA-seq of Mus musculus: lymph nodes

city: New Haven

first: Steven

last: Kleinstein

state: Ct

address: 300 George St.

department: Pathology

institute: Yale University

data_processing:
Single cell feature counts for a single library were generated using `cellranger count` from Cell Ranger v3.1.0
Genome_build: mm10
Supplementary_files_format_and_content: matrix.mtx.gz contains a gene expression count matrix stored in the Market Exchange Format for sparse matrices
Supplementary_files_format_and_content: features.tsv.gz contains feature information that correspond to row indices of matrix.mtx.gz
Supplementary_files_format_and_content: barcodes.tsv.gz contains cellular barcodes that correspond to column indices of matrix.mtx.gz

source_name: Lymph nodes

extract_protocol:
Total antibody-secreting cells (CD19+ CD4- IgDlo Fas+ CD38lo CD138+) were bulk sorted from draining lymph nodes.
Libraries were prepared using 10x Genomics' Chromium Next GEM single cell 5' gene expression workflow

treatment_protocol: Intramuscular injection of 10 microgram of the receptor-binding domain (RBD) of the spike protein was administered on Day 0. Intramuscular injection of 5 microgram of the whole spike protein was administered on Day 14, and then again on Day 24. Draining lymph nodes were harvested on Day 29.

tag: strain

tag: age

value: 10 weeks

tag: Sex

value: female

value: Iliac and inguinal lymph nodes


title: GlcNAc-DL1-3

status: Public on Jun 09 2020

type: SRA

city: Gainesville

first: Lin

last: Zeng

state: FL

address: 1395 Center Drive

department: Oral Biology

country: USA

institute: University of Florida

library_source: transcriptomic

data_processing:
Step 1: FASTQ Groomer
Step 2: FASTQ Quality Trimmer
Step 3: Cutadapt removing GATCGGAAGAGCACACGTCTGAACTCCAGTCAC
Step 4: Map with Bowtie for Illumina
Step 5: sort
Step 6: htseq-count (see our publication for details)
Genome_build: NC_004350.2
Supplementary_files_format_and_content: Counts obtained from step 6: htseq-count, listed along side each open reading frame

molecule: total RNA

organism: Streptococcus gordonii

source_name: Bacterial batch culture

growth_protocol: Bacterial strains UA159 and DL1 were first cultivated overnight, each with 3 individual cultures, on a TV medium supplemented with 20 mM of glucose, GlcN, or GlcNAc, then diluted at 1:20 ratio into a fresh TV medium containing the same carbohydrate. When the optical density (OD600) of an individual culture reached around 0.5, a portion of the culture was placed on ice temporarily before use. The rest of the culture was harvested by centrifugation, treated with the RNAprotect Bacteria Reagent (Qiagen, Germantown, MD) and stored at -80°C. Once all cultures were ready on ice, UA159 and DL1 grown on the same carbohydrates were mixed at 20:1 ratio based on their OD600, before diluted again at 1:50 ratio into the same medium. After the mixed-species co-culture reached OD600 ≈ 0.5, cells were harvested as before and stored at -80°C.

extract_protocol:
Total RNA was extracted from bacterial cells using the RNeasy Mini kit (Qiagen), with chromosomal DNA removed using a DNase I kit (Qiagen). To remove 16S and 23S rRNAs, 10 µg of total RNA was treated using the MICROBExpressTM Bacterial mRNA Enrichment Kit
cDNA libraries were created from 100 ng enriched mRNA samples using the NEBNext Ultra Directional RNA Library Prep kit for Illumina and NEBNext multiplex Oligos for Illumina (New England Biolabs, Ipswich, MA) following instructions from the supplier.
RNA-Seq: single-end short reads were obtained from a set of bar-coded cDNA libraries

treatment_protocol: Cells were harvested by centrifugation at 4°C for 5 min, treated with bacterial RNAprotect Bacteria Reagent (Qiagen), and immediately stored at -80°C.

tag: parental strain

tag: genotype

value: Wild type

tag: media

value: TV-GlcNAc


title: BC3 2

city: Shanghai

first: Peizhan

last: Chen

country: China

institute: Ruijin Hospital North

data_processing:
Cell Ranger
R preprocessing for doublets removal
mitochondrial genes proportion (≦ 10%) and the numer of detected genes (≧300 features)
Genome_build: GRCH38
Supplementary_files_format_and_content: Matrix table with raw UMI counts for every gene and every single cell in the specific patients

molecule: polyA RNA

organism: Homo sapiens

source_name: Osteosarcoma patients

extract_protocol:
The tissue pieces were digested with 2 mL of GEXSCOPETM tissue dissociation solution (Singleron Bio Com, Nanjing, China) at 37 °C for 15 min with sustained agitation. After digestion, the samples were filtered through 40-µm sterile strainers and centrifuged at 1,000 rpm for 5 min. Then the supernatants were discarded, and the cell pellets were suspended in 1 mL phosphate-buffered saline (PBS; HyClone). To remove red blood cells, 2 mL of GEXSCOPETM red blood cell lysis buffer (Singleron) was added, and cells were incubated at 25 °C for 10 min. The solution was then centrifuged at 500 g for 5 min and resuspended in PBS.  Single cells were encapsulated into emulsion droplets using the Chromium Controller (10 x Genomics).
The scRNA-seq libraries were constructed using the Chromium Single Cell 3’ Library, Gel Bead & Multiplex Kit (10x Genomics)

tag: tumor type

value: Osteoblastic


title: HOXB9

city: Durham

first: Bryan

middle: J

last: Venters

state: NC

address: 6 Davis Drive

institute: EpiCypher

description: CUT&RUN HOXB9 500K Nuclei

library_source: genomic

data_processing:
Library strategy: CUT&RUN
Base-calling: illumina software
Adapter Trimming: TBD
Alignment: TBD
Filtering: TBD
Peak Calliing: TBD
Genome_build: hg19
Supplementary_files_format_and_content: PCRP_Metadata.xlsx: contains one excel sheet

molecule: genomic DNA

source_name: K562 human immortalised myelogenous leukemia cell line

growth_protocol: K562 cells were maintained (1–10 × 10^5 cell/ml) in T25 flasks in IMDM + 10% FBS  +  1% Pen/Strep

extract_protocol:
Nuclei were extracted by incubating cells on ice for 10 min in Nuclei Extraction buffer (NE: 20 mM HEPES–KOH, pH 7.9; 10 mM KCl; 0.1% Triton X-100; 20% Glycerol; 0.5mM spermidine; 1x complete protease inhibitor [Roche # 11836170001]), collecting by centrifugation (600 g, 3 min, 4ºC), discarding the supernatant, and resuspending at [100 µl / 500K nuclei] sample in NE buffer. CUTANA protocol v1.5.1; optimized version of PMID: 29651053
NEBNext Ultra II DNA Library Prep kit (New England Biolabs #E7645S)
Cleavage under targets and Release using nuclease

treatment_protocol: Native cells used for Nuclei extraction

tag: cell line

tag: antibody


title: THP-1 cells, siFli1 3

type: RNA

city: Boston

first: Boston University

last: Microarray and Sequencing Resource

state: MA

address: 72 East Concord Street, E631

department: Microarray and Sequencing Resource

institute: Boston University

description: Gene expression data from THP-1 cells treated with siFli1.

hyb_protocol: The labeled, fragmented DNA was hybridized to the Affymetrix Human Gene 2.0 ST Array for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.

scan_protocol: After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus.

data_processing: Raw Affymetrix CEL files were normalized to produce Entrez Gene-identifier-specific expression values using the implementation of the Robust Multiarray Average (RMA) in the affy Bioconductor package (version 1.36.1), using R version 2.15.1 and the Brainarray hugene20sthsentrezgcdf R package (version 23.0.0).

label: biotin

source_name: THP-1 cells

growth_protocol: THP-1 cells were grown in RPMI media.

extract_protocol: Cells were lysed using TRIzol and RNA was then extracted using Zymo Research kit and integrity analysed  using Agilent Bioanalyzer. Purity of the RNA samples was confirmed using a NanoDrop spectrophotometer.100 ng of high quality RNA (RIN >9.0) with Biotin labeling was performed using the WT Plus reagent kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol.

treatment_protocol: Fli1 expression was inhibited using siRNA. Cells were then converted to M0 macrophages 24 h later using 10 ng/ml PMA (Phorbol 12-myristate 13-acetate) for 4 hours, then media changed and cells treated with 100 ng/ml M-CSF (macrophage colony stimulating factor) for 48 hours.

tag: sirna

value: Fli1 downregulation


title: FOXA2

description: CUT&RUN FOXA2 500K Nuclei


title: HOXB5

description: CUT&RUN HOXB5 500K Nuclei


title: WT-16 15948X1

city: Salt Lake City

first: Jordan

middle: A

last: Berg

state: UT

address: 15 N Medical Drive East, Rm 5520

department: Biochemistry

institute: University of Utah

description:
WT-16
total RNA (post-rRNA subtraction)

data_processing:
CUTADAPT v 1.16: CUTADAPT -j $NCPU -O 6 -m 20 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA  -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT  -o $OUT.1.fq -p $OUT.2.fq $OUT.clump1.fq.gz $OUT.clump2.fq.gz
FASTQ: $FASTQC -T $NCPU -f fastq $OUT.1.fq
: $FASTQC -T $NCPU -f fastq $OUT.2.fq
STAR: $STAR --genomeDir $INDEX --readFilesIn $OUT.1.fq $OUT.2.fq --runThreadN $NCPU --twopassMode Basic --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM --outWigType bedGraph --outWigStrand Unstranded
RSEM: $RSEM --paired-end -p $NCPU --alignments --strandedness reverse --no-bam-output Aligned.toTranscriptome.out.bam $RSEM_INDEX $OUT
featureCounts: $FEATCOUNT -T $NCPU -p -s 2 --largestOverlap -a $GTF -o $OUT.counts $OUT.bam
: $FEATCOUNT -T $NCPU -p -s 2 --largestOverlap -a $GTF -o $OUT.biotypes -g gene_biotype $OUT.bam
Samtools: $SAMTOOLS index $OUT.bam
: $SAMTOOLS idxstats $OUT.bam | sort -V > $OUT.idxstats
Genome_build: Ensembl Mus musculus Build GRCm38 version 96
Supplementary_files_format_and_content: tab-delimited file

organism: Mus musculus

source_name: Mouse heart tissue

extract_protocol:
RNA was isolated from ventricular tissue samples usingmiRNeasyMini Kit. Total RNA samples (100-500 ng) were hybridized with Ribo-Zero Gold to substantially deplete cytoplasmic and mitochondrial rRNA from the samples.
Stranded RNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded Total RNA Library Prep Gold kit (20020598) with TruSeq RNA UD Indexes (20022371).  Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824).  Individual libraries were normalized to 1.30 nM in preparation for Illumina sequence analysis. Sequencing libraries (1.3 nM) were chemically denatured and applied to an Illumina NovaSeq flow cell using the NovaSeq XP chemistry workflow (20021664).  Following transfer of the flowcell to an Illumina NovaSeq instrument, a 2 x 51 cycle paired end sequence run was performed using a NovaSeq S1 reagent Kit (20027465).

value: wild type

tag: timepoint

value: 16 weeks post-knockout


title: BC3 1


title: GlcNAc-DL1-2


title: GlcNAc-DL1-1


title: BC2 12


title: BC3 5


title: GlcN-DL1-3

value: TV-GlcN


title: STAT1

description: CUT&RUN STAT1 500K Nuclei


title: BC3 4


title: BC2 11


title: THP-1 cells, siFli1 1

label_protocol: Biotin labeling was performed using the WT Plus reagent kit (Affymetrix, Santa Clara, CA) according to the manufacturer's protocol.


title: THP-1 cells, Scrambled control 3

description: Gene expression data from THP-1 cells treated with scrambled control.

value: scrambled control

value: control


title: GlcN-DL1-2


title: MAX

description: CUT&RUN MAX 500K Nuclei


title: BC2 10


title: GlcN-DL1-1


title: THP-1 cells, siFli1 2


title: BXL0124, Bisulfite-seq

city: Piscataway

first: Nanjoo

last: Suh

state: NJ

address: 164 Frelinghuysen Road

department: Chemical Biology

institute: Rutgers, The State University of New Jersey

description: vitamin D analog treatment

data_processing:
Illumina standard procedure for basecalling
The reads were aligned using Bismark software (version 0.15.0)
CpG sites were counted and clustered into Differentially Methylated Regions (DMRs) using DMRfinder (version 0.1)
Genomic annotation was performed with ChIPseeker in R
Genome_build: hg19
Supplementary_files_format_and_content: Tab delimited tables of counts of methylation and unmethylation for each CpG site

source_name: MCF10DCIS mammospheres

growth_protocol: MCF10DCIS cells grown in mammosphere culture medium for 5 days

extract_protocol:
Cells were dissociated with Accutase and DNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA)
The DNA samples were prepared using an Agilent SureSelect Human Methyl-seq Target Enrichment System (Agilent Technologies, Santa Clara, CA)

treatment_protocol: Mammospheres were treated with DMSO, 1,25D3 and BXL0124

tag: condition

value: mammospheres

tag: duration of treatment

value: 5 Days

tag: agent

value: vitamin D analog


title: BC2 9


title: 1,25D3, Bisulfite-seq

description: vitamin D treatment

value: vitamin D


title: BC2 8


title: Glc-DL1-3

value: TV-glucose


title: control, Bisulfite-seq

description: Control treatment

value: Control


title: THP-1 cells, Scrambled control 1


title: Glc-DL1-2


title: BC2 7


title: MEN1

description: CUT&RUN MEN1 500K Nuclei


title: Glc-DL1-1


title: Pleural_New_rep8

city: River Forest

first: Robert

last: Calin-Jageman

state: IL

address: 7900 West Division

department: Psychology

institute: Dominican University

description: Sample z284

hyb_protocol: We used the Aplysia Tellabs Array (ATA: GEO: GPL18666) to characterize changes in gene expression in 3 phases of memory (New, Forgotten, and Savings).  The array was designed using the UniGene clustering of Aplysia ESTs (build 9, July 2011, ftp://ftp.ncbi.nih.gov/repository/UniGene/Aplysia_californica/) and includes 2-3 probes designed for each of the 24,702 distinct EST clusters identified by Unigene, 15 Aplysia mRNAs deposited in GenBank between the Unigene build and the design of the array, and 1,432 orphaned probes from two previous Aplysia microarray designs (the Aplysia Discovery Array (GEO:GPL3635) and the updated Aplysia Annotated Array (GEO: GPL13815/GPL17112)).  Thus, the ATA design includes 26,149 distinct probes representing all known sources of Aplysia californica ESTs and mRNAs at the time of design (January 2012).   Based on estimates from previous microarray designs (Moroz et al. 2006), the ATA should cover >50-60% of all neurally-expressed transcripts.  Complete details on the ATA design are in Herdegen et al. (Herdegen et al. 2014b). Microarray processing was completed by Mogene Inc. (St. Louis, MO).  A two-color approach was used with each array hybridized to a sample from a trained or untrained animal.  In half of cases, trained samples were hybridized with Cy3 and controls to Cy5; the other half we dye-swapped. Sample integrity was determined by Bioanalyzer RNA 6000, Pico total RNA protocol.  300ng of total RNA was amplified and labeled with Cy3 or Cy5 using the Agilent Quick Amp Two-Color Labeling Kit. Dye incorporation and yield was determined by Nanodrop.  Samples were hybridized to the microarray slide at 65C and 10rpm for 17 hours.  Slides were scanned on an Agilent C scanner at 3µm resolution.  Data was extracted using Agilent Feature Extraction software, v. 11.5.  All labeling and post-labeling processing was carried out in an ozone regulated environment, monitored at < 5ppb.

scan_protocol: Slides were scanned on an Agilent C scanner at 3um resolution.  Data was extracted using Agilent Feature Extraction software, v. 11.5.

data_processing: Microarray data was analyzed using limma (Smyth 2005; Ritchie et al. 2015a) from the Bioconductor suite of tools (Gentleman et al. 2004) for R (Ihaka and Gentleman 1996).   Median expression values were analyzed (Zahurak et al. 2007).  These were corrected for background using the normexp+offset algorithm recommended for Agilent microarrays by Ritchie et al. (Ritchie et al. 2007).  An offset of 30 was selected based on inspection of MA Plots (Supplemental Figure 2).  Expression was then normalized using the loess function (Smyth and Speed 2003).  Where multiple probes were used to measure the same EST or mRNA, these were averaged.  Finally, trained and control expression were compared using an empirical Bayes-moderated t-test (Smyth 2004).  Statistical significance was calculated using Benjamini-Hochberg correction for multiple comparisons to maintain a 5% overall false-discovery rate (Benjamini and Hochberg 1995).

organism: Aplysia californica

source_name: New

label_protocol: A two-color approach was used with each array hybridized to a trained (Cy5) or untrained (Cy3) sample.  Sample integrity was determined by Bioanalyzer RNA 6000, Pico total RNA protocol.  300ng of total RNA was amplified and labeled with Cy3 or Cy5 using the Agilent Quick Amp Two-Color Labeling Kit. Dye incorporation and yield was determined by Nanodrop.

growth_protocol: Animals (75-125g) were obtained from the RSMAS National Resource for Aplysia (Miami, FL) and maintained at 16o C in one of two 90-gallon aquariums with continuously circulating artificial sea water (Instant Ocean, Aquarium Systems Inc.).  Animals were separately housed in rectangular colanders (11.5cm wide, 16.5cm long, 7.3cm deep), fed dried seaweed twice a week, and maintained on a 12 hr light-dark cycle.  2 days prior to any experimental testing, animals were fed to satiation and then food deprived until after the 1-day post-tests (Levenson, Byrne, & Eskin, 1999; Wainwright, Zhang, Byrne, & Cleary, 2002).  Food deprivation was resumed the day before the 7-day post-tests.  To control for batch/shipment effects, animals from at least 2 different shipments were used for each experiment.

extract_protocol:
We compared gene expression from pleural ganglia on the trained vs. untrained side of the animal.  Samples from two animals trained on opposite sides were pooled.  This was done to control for lateralized gene expression.
To analyze transcription, pleural ganglia RNA was isolated immediately after the 8-day post-tests.  Briefly, animals were anesthetized with an injection of isotonic MgCl2 (50% of body weight), and an incision was then made along the ventral midline to expose the CNS.  As dissection can alter gene expression (Alberini et al. 1994), we extracted ganglia rapidly (< 5 minutes per animal) and transferred them immediately to Trizol (Invitrogen, Carlsbad CA) for homogenization.
Tissue was homogenized using the Bullet Blender (NextAdvance, Averill Park, NY) and RNA extracted using Direct-Zol Mini RNA Kit (Zymo, Irvine, CA). Quantity and quality of RNA was assessed using the NanoDrop 1000 (Thermo Scientific, Wilmington, DE).

treatment_protocol:
A one-day long-term sensitization training protocol was used (Bonnick et al., 2012) .  Training consisted of 4 rounds of noxious shock applied at 30 minute intervals to one side of the body with a hand-held electrode.  Each round of shock consisted of 10 pulses (60Hz biphasic) of 500ms duration at a rate of 1hz and an amplitude of 90mA.  Side of training was counterbalanced.  This training protocol produces memory that is strongly expressed for several days but which fades in most animals within 1 week (Perez et al., 2018).
The savings- and forgotten-memory groups received long-term sensitization training immediately after pre-tests, on the first day of the protocol.   In contrast, the new-memory group initially received sham training.  This consisted of the same procedure but the constant-current stimulator was set to deliver 0mA of current.  Animals were otherwise handled in the same way and, in general, were run mixed with batches of animals from the other conditions.  For the new-memory group, real sensitization training was finally applied after the 7-day tests, 1 day prior to harvesting tissue.
Reminders to elicit savings
To elicit savings, animals in the savings group received a reminder shock (Perez et al., 2018; Philips et al., 2006).  The reminder was delivered 7 days after training, when most animals show essentially no remaining sensitization memory (< 25% increase relative to pre-test).  The reminder consisted of two moderate shocks (60Hz biphasic DC pulse for 2s at 20ma of constant current) applied to the midline of the tail with a 15-minute rest between the shocks.  The reminder produces short- but not long-term sensitization in naïve animals.  In previously trained animals the reminder reveals a long-lasting unilateral savings memory, with T-SWR durations increasing for at least 1 day after the reminder but only on the previously trained side (Perez et al., 2018).
Animals in the forgetting group received a sham reminder, where the same protocol was applied but the constant-current stimulator was dialed to deliver 0mA of current.
Animals in the new-memory group did not receive a reminder or a sham reminder, but instead received their sensitization training while the other groups received reminders.

tag: tissue

value: pleural ganglia

tag: phase of memory

value: New

tag: sensitization

value: untrained

source_name: Control

value: trained


title: THP-1 cells, Scrambled control 2


title: Pleural_Forgotten_rep7

description: Sample z295

source_name: Forgotten

value: Forgotten


title: Pleural_New_rep7

description: Sample z283


title: Pleural_Forgotten_rep8

description: Sample z269


title: BC2 6

value: Osteoblastic


title: BXL0124

description: vitamin D analog treatment

data_processing:
Illumina standard procedure for basecalling
RNA-seq reads were mapped to human reference genome with Hisat2 software
Cufflinks was used to assemble the transcript products and calculate the fragment abundance
Cuffdiff was used to quantify transcripts of genes differentially expressed among the three treatment conditions (DMSO, 1,25D3 and BXL0124)
Genome_build: hg19
Supplementary_files_format_and_content: Tab delimited tables with FPKM values for each sample

extract_protocol:
Cells were dissociated with Accutase and RNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA)
RNAseq libraries were prepared using Illumina RNA Library Prep Kit v2 according to manufacturer's user guide with 400 ng of RNA as input


title: Pleural_Savings_rep8

description: Sample z261

source_name: Savings

value: Savings


title: Pleural_Savings_rep7

description: Sample z260


title: Pleural_Savings_rep6

description: Sample z256


title: mutant pp1r3-2  topp4-1 3

city: FuZhou

first: Jing

last: Zhang

state: Fujian Province

address: No.15 Shangxiadian Road, Cangshan District, Fuzhou City, Fujian Province, China

institute: Fujian Agriculture and Forestry University

description: processed data_gene_count.xls [r3g39_3]

data_processing:
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.
Genome_build: Arabidopsis_thaliana_Ensemble_44
Supplementary_files_format_and_content: Excel table contains the gene readcount of each gene in each sample and the basic information of the gene.

organism: Arabidopsis thaliana

source_name: leaf

growth_protocol: Arabidopsis (Columbia, Col) was used in the present study. And the plants used for RNA-seq were grown in a greenhouse under a 16-h light/8-h dark photoperiod at 23℃.

extract_protocol:
Novogene uses standard extraction methods to extract RNA from tissues or cells, and then strictly controls the RNA samples. RNA quantification and qualification ①RNA degradation and contamination was monitored on 1% agarose gels. ② RNA purity was checked using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA) . ③ RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system

treatment_protocol: Normal growth conditions, wild type (Col) as a control

tag: ecotype

value: leaf

value: 21 day after germination

tag: genotype/variation

value: mutant pp1r3-2  topp4-1


title: Pleural_Forgotten_rep6

description: Sample z294


title: 1,25D3

description: vitamin D treatment


title: mutant pp1r3-2  topp4-1 2

description: processed data_gene_count.xls [r3g39_2]

tag: age


title: control

description: Control treatment


title: BC2 5


title: BC2 4


title: BC2 3


title: topp septuple mutants topp-7m 3

description: processed data_gene_count.xls [topp7m_3]

value: topp septuple mutants topp-7m


title: Pleural_Forgotten_rep5

description: Sample z275


title: BC2 2


title: Pleural_Savings_rep5

description: Sample z242


title: Pleural_New_rep5

description: Sample z276


title: topp septuple mutants topp-7m 2

description: processed data_gene_count.xls [topp7m_2]


title: mutant topp4-1 3

description: processed data_gene_count.xls [g39_3]

value: mutant topp4-1


title: mutant pp1r3-2  topp4-1 1

description: processed data_gene_count.xls [r3g39_1]


title: mutant topp4-1 2

description: processed data_gene_count.xls [g39_2]


title: Pleural_New_rep6

description: Sample z288


title: mutant pp1r3-2 3

description: processed data_gene_count.xls [r3_3]

value: mutant pp1r3-2


title: BC2 1


title: topp septuple mutants topp-7m 1

description: processed data_gene_count.xls [topp7m_1]


title: ER- Luminal Cells Sox9 KO2

city: Bronx

first: John

middle: Robert

last: Christin

state: NY

address: 1301 Morris Park Avenue, Room 108/114

department: Cell Biology

institute: Albert Einstein College of Medicine

hyb_protocol: GeneChip™ WT PLUS

scan_protocol: Affymetrix Genechip Scanner 3000 7G

data_processing: TAC 4.0 default settings

label: Biotin

source_name: ER- Luminal Cells

label_protocol: GeneChip™ WT PLUS

extract_protocol: Trizol LS

value: Sox9 KO


title: Pleural_Forgotten_rep4

description: Sample z277


title: ER- Luminal Cells Sox9 KO1


title: ER- Luminal Cells Sox9 KO3


title: R84S_7_C

city: Philadelphia

first: Elizabeth

last: White

state: PA

address: 3610 Hamilton Walk, 203C Johnson Pavilion, Mail Code 6114

department: Otorhinolaryngology

institute: University of Pennsylvania

data_processing:
Raw image data file from high-throughput sequencing was transformed to Sequenced Reads(called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling).
Sequenced reads were filtered to remove adapters and low-quality reads
Clean reads were mapped to hg19 using STAR (v2.5). STAR used  the method of Maximal Mappable Prefix (MMP).
HTSeq v0.6.1 was used to count the read numbers mapped of each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis between conditions/groups was performed using the DESeq2 R package (2_1.6.3).
Genome_build: hg38
Supplementary_files_format_and_content: gene expression quantification table, calculated all genes' FPKM

source_name: Primary human forekin keratinocytes

growth_protocol: Primary human keratinocytes stably harboring retroviral expression vectors were grown to 30% confluence in keratinocyte serum-free medium with 0.5 ug/ml puromycin

extract_protocol:
RNA was isolated with Qiagen RNeasy mini kit in White Laboratory and polyA RNA was isolates using oligo dT beads at Novogene Corporation
RNA libraries were prepared for sequencing using standard Illumina protocols at Novogene Corporation

tag: retrovirus

value: Tranduced with HPV18 E7 R84S retrovirus (MSCV-P C-FlagHA 18E7 R84S)

value: Primary human foreskin keratinocytes selected with puromycin post retroviral transduction


title: wt18_7_B

value: Tranduced with HPV18 E7 retrovirus (MSCV-P C-FlagHA 18E7)


title: wt18_7_C


title: R84S_7_B


title: ER- Luminal Cells WT3

value: WT


title: Pleural_New_rep4

description: Sample z268

value: pleural ganglia

value: New

value: Control


title: Pleural_New_rep3

description: Sample z234

value: untrained

value: trained


title: Pleural_New_rep2

description: Sample z238


title: Pleural_Savings_rep4

description: Sample z241

value: Savings


title: ER- Luminal Cells WT2


title: Pleural_Forgotten_rep2

description: Sample z280

value: Forgotten


title: ER- Luminal Cells WT1


title: Luminal mammary  epithelial cells, Sox9-GFP_Lo_3

source_name: Luminal Sox9-GFP low mammary epithelial cells

extract_protocol: Qiagen Minielute RNA

tag: cell type

value: Luminal mammary epithelial cells

tag: facs-sorted cells


title: Luminal mammary  epithelial cells, Sox9_GFP_Hi_3

source_name: Luminal Sox9-GFP high mammary epithelial cells


title: mutant pp1r3-2 2

description: processed data_gene_count.xls [r3_2]

value: leaf

value: 21 day after germination


title: Pleural_Savings_rep3

description: Sample z239


title: Pleural_Forgotten_rep3

description: Sample z278


title: mutant topp4-1 1

description: processed data_gene_count.xls [g39_1]

value: mutant topp4-1


title: R84S_7_A


title: Pleural_Savings_rep2

description: Sample z237


title: wt18_7_A


title: mutant pp1r3-2 1

description: processed data_gene_count.xls [r3_1]

value: mutant pp1r3-2


title: cont_7_C

value: Tranduced with control retrovirus (MSCV-IP N-HA empty)


title: wild type Col 3

description: processed data_gene_count.xls [Col_3]

value: wild type


title: Pleural_New_rep1

description: Sample z292


title: cont_7_A

value: Primary human foreskin keratinocytes selected with puromycin post retroviral transduction


title: wild type Col 1

description: processed data_gene_count.xls [Col_1]


title: wild type Col 2

description: processed data_gene_count.xls [Col_2]


title: PLC/PRF/5 sgPPARD

city: Beijing

first: Yanying

last: Wang

address: 15 Datun Road, Chaoyang District,Beijing 100101, China

institute: Institute of Biophysics, Chinese Academy of Sciences

data_processing:
The sequencing data was filtered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) 60 was applied to align the clean reads to the reference coding gene set，then expression level of gene was calculated by RSEM (v1.2.12). The heatmap was drawn by pheatmap (v1.0.8) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05. To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) by Bonferroni.
Genome_build: hg18
Supplementary_files_format_and_content: txt format including Gene id, gene symbol, diffexp_log2fc_PLCshSCR-vs-PLCshPPARD, diffexp_possiondis_fdr__PLCsgCtrl-vs-PLCsgPPARD, diffexp_possiondis_pvalue__PLCsgCtrl-vs-PLCsgPPARD

source_name: sorted cells

growth_protocol: Human hepatocellular carcinoma (HCC) cell lines were obtained from ATCC and maintained in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin G, and 100 U/ml streptomycin (Invitrogen, NY, USA).

extract_protocol:
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25μL~100μL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and pair end 100 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

treatment_protocol: HCC PLC/PRF/5 cells were depleted PPARD using CRISPR/Cas9 mediated gene editing, sgRNA sequences were cloned into lentiCRISPRv2-puro plasmid. LentiCRISPRv2, pVSVg (8454), and psPAX2 (12260) were transfected into 293T cells to generate CRISPR/Cas9 lentivirus.

value: HCC cultured cells

tag: treatment

value: transfected with sgRNA against PPARD


title: PLC/PRF/5  sgCtrl

value: transfected with  control sgRNA

tag: cell line


title: Luminal mammary  epithelial cells, Sox9-GFP_Lo_2


title: Luminal mammary  epithelial cells, Sox9_GFP_Hi_2


title: Pleural_Forgotten_rep1

description: Sample z281

value: pleural ganglia


title: Luminal mammary  epithelial cells, Sox9-GFP_Lo_1


title: Pleural_Savings_rep1

description: Sample z235

value: Control


title: BRL_3A_siRNA_2

status: Public on Jun 08 2020

first: dongsheng

last: xiao

address: No.151 Libing Road, Zhangjiang Hi-Tech Park Pudong, Shanghai 201203, P.R. China

institute: Shanghai Biotechnology Corporation

description: Agilent Whole Rat Genome Oligo Microarray 4x44K

hyb_protocol: Each slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat.# 121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat.# 5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions。

scan_protocol: Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=3μm, PMT 100%, 20bit.

data_processing: Data were extracted with Feature Extraction software 12.0 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, limma packages in R.

organism: Rattus norvegicus

source_name: BRL-3A cell line

label_protocol: Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat.# 5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat.# 74106, QIAGEN, GmBH, Germany)

growth_protocol: The BRL-3A rat liver cell line （ATCC） were cultured in glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA) at 37°C with humidified air and 5% CO2

extract_protocol: Total RNA was extracted and purified using miRNeasy Mini Kit（Cat#217004，QIAGEN, GmBH, Germany) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).

treatment_protocol: All of siRNAs were transfected into cells applying lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to manufacture's protocol for 48h then haverst cells. The cultured BRL-3A cells were collected and added to trizol for lysis after storage at -80.

tag: transfection

value: siRNA lnc19959.2

value: Liver

tag: time point


title: BRL_3A_siRNA_3


title: BRL_3A_siRNA_4


title: CB_LR

city: Wuhan

first: ying

last: chen

address: 1277 Jiefang Avenue, Wuhan, Hubei Province

department: Institute of Hematology

institute: Union Hospital，Tongji Medical College, Huazhong University of Science and Technology

data_processing:
In order to guarantee the data quality which was used to analysis, the useful Perl script was used to filter the original data (Raw Data).
The reference genomes and the annotation file were downloaded from ENSEMBL database (http://www.ensembl.org/index.html). Bowtie2 v2.2.3 was used for building the genome index, and Clean Data was then aligned to the reference genome using HISAT2 v2.1.0. HISAT2 is the successor to TopHat2, which uses a modified BWT algorithm to convert reference genomes to index for faster speed and fewer resources.
The IGV (Integrative Genomics Viewer) was used to view the mapping result by the Heatmap, histogram, scatter plot or other stytle.
Reads Count for each gene in each sample was counted by HTSeq v0.6.0, and FPKM (Fragments Per Kilobase Millon Mapped Reads) was then calculated to estimate the expression level of genes in each sample.
Genome_build: Homo_sapiens.GRCh38.91.chr
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: CD34+ human cord blood-derived cells

extract_protocol:
Total RNA was extracted using Trizol reagent (Takara, Japan) following the manufacturer's procedure. The libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: modulating agent

value: LY plus Rapa

value: hUCB CD34+ cells


title: CB_SR1


title: CB_Uncultured


title: CB_HGFs

value: DMSO


title: cont_7_B


title: BRL_3A_siRNA_1


title: BRL_3A_NC_4

value: NC


title: BRL_3A_NC_3


title: CD133_KO2

city: Tianjin

first: Jie

last: Li

address: Wei Jin Road Nankai District No. 94

department: Department of Cell Biology and Genetics

institute: Nankai University

data_processing:
The RNA-seq reads were aligned to the human reference genome hg19 using Hisat2
Sequenced reads were trimmed for adaptor sequence using Trimmomatic
the read counts were adjusted for each sequenced library by featureCounts R package through one scaling normalized factor
Genome_build: hg19
Supplementary_files_format_and_content: CPM

source_name: Embryonic stem cells

growth_protocol: WA26 human embryonic cells were routinely maintained in undifferentiated state in E8 medium (A1517001, Life technologies) on Matrigel (356230, BD Bioscience) coated tissue culture plates with daily medium feeding and passaged every 3-4 days with 0.5 mM EDTA in PBS at 1:20 ratio with Rocki (sc-281642A, Santa Cruz) for maintenance

extract_protocol: Sequencing libraries were generated  by using TruePrep DNA Library Prep Kit V2 for Illumina® (TD503-02, Vazyme Biotech)

value: WA26  human embryonic cells

tag: passages

tag: genotype


title: CD133_KO1


title: CD133_WT2


title: BRL_3A_NC_2


title: BRL_3A_NC_1


title: CD133_WT1


title: Retina-Treated, 2349

city: Spokane

first: Mike

last: Gibson

state: Washington

address: 412 E Spokane Falls Blvd

department: Pharmacotherapy

institute: Washington State University

data_processing:
HTSeq v 0.6.1 was used to count the read numbers mapped of each gene, FPKM of each was calculated based on the lengh of the genes and reads counpt mapped to this gene
DESeq2. Differential expression analysis between two conditions / groups was performed using the DESeq2 R package (2_1.6.3). DESeq2 provided statistical routines for determing differential expression
Differential expression was also performed using the edgeR R package (3.16.5)
Genome_build: mm10
Supplementary_files_format_and_content: Matrix Table with readcount for each sample

source_name: Retina

extract_protocol:
Retinas were removed, flash frozen on dry ice, and RNA was harvested using Qiazol (Qiagen) reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: retina

value: 10-12 weeks old

value: VGB 140/mg/kg/day


title: Retina-Treated, 2429

tag: tissue


title: Retina-Treated, 2428


title: Retina-Treated, 2347


title: Retina-Treated, 2344


title: Retina-Untreated, 835

value: Vehicle


title: Spleen monocytes rep2

city: Winston-Salem

first: Yong

last: Lu

address: 575 N. Patterson Ave

department: Dept of Microbiology and Immunology

institute: wake forest school of medicine

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Rat Gene 2.0. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G3000 7G.

data_processing: The data were analyzed with RMA using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

source_name: Spleen monocytes

label_protocol: Samples with intact 18S and 28S ribosomal RNA bands with RIN >8.5 were processed for microarray analysis, which was performed with a Mouse Gene 2.0 ST Kit at the Case Western Reserve University Genomic Core.

growth_protocol: The cells were sorted from spleen tissue or tumor tissue.

extract_protocol: Total RNA was extracted with the RNeasy Mini kit (Qiagen).

treatment_protocol: Mice were treated with Th9 cells, and 10 days after Th9 transfer, tumor tissue and spleens were harvested and dissociated for cell sorting. CD11b+CD11c–Ly6Chi inflammatory monocytes were flow-sorted.

value: Spleen


title: Retina-Untreated, 832


title: Retina-Untreated, 2345


title: Luminal mammary  epithelial cells, Sox9_GFP_Hi_1

value: Luminal mammary epithelial cells


title: Tumor monocytes rep3

source_name: Tumor monocytes

value: Tumor


title: Spleen monocytes rep1


title: Spleen monocytes rep3


title: Retina-Untreated, 1582


title: Tumor monocytes rep1


title: Tumor monocytes rep2


title: Retina-Untreated, 1580


title: cd8_cd5hi_4

city: Bethesda

first: Apratim

last: Mitra

state: MD

address: 6 Center Dr Building 6B Room 2B206

department: Division of Developmental Biology

institute: NICHD

data_processing:
The 101 bp paired-end reads were aligned using STAR version 2.5.2a and reads quantified with featureCounts version 1.4.6-p3. Differential expression analysis was performed using DESeq2(71). We used a false discovery rate cutoff of < 1% to define statistical significance.
Genome_build: mm10
Supplementary_files_format_and_content: Raw counts table: 24421 rows x 16 sample columns + geneid column with gene symbols.

source_name: T cells_CD8+CD5hi

extract_protocol:
RNA was extracted from FACS-sorted T cells using TRIzol reagent (Invitrogen) followed by purification and DNase treatment with RNeasy columns (Qiagen) and submitted for library construction.
Library construction was performed using the TruSeq RNA sample preparation kit version 2 or TruSeq stranded mRNA sample preparation kit (Illumina).

treatment_protocol: Total lymph node cells from C57BL/6 mice were stained with fluorochrome conjugated antibodies against CD4, CD8, CD5, CD25, CD62L and CD44. Sorted populations consisted of the 1/3 of CD25-CD62Lhi CD44lo CD4+ or CD8+ T cells expressing the highest surface levels of CD5 (CD5hi) or the 1/3 of CD25-CD62Lhi CD44lo CD4+ or CD8+ T cells expressing the lowest surface levels of CD5(CD5lo).

tag: mouse strain

value: Total lymph node cells

tag: cell subtype


title: cd8_cd5hi_2


title: cd8_cd5lo_4

source_name: T cells_CD8+CD5lo


title: cd8_cd5hi_1


title: cd8_cd5lo_3


title: cd8_cd5lo_2


title: cd4_cd5hi_4

source_name: T cells_CD4+CD5hi


title: cd4_cd5hi_3


title: cd4_cd5lo_4

source_name: T cells_CD4+CD5lo


title: cd4_cd5hi_2


title: cd4_cd5hi_1


title: cd4_cd5lo_3


title: cd4_cd5lo_2


title: cd4_cd5lo_1


title: CD4+lymphocyte from litter mate_3

city: Daejeon

first: Yukyung

last: Jun

address: 245 Daehak-ro, Yuseong-gu

department: Center for Supercomputing Applications

country: South Korea

institute: Korea Institute of Science and Technology Information

description: CD4+lymphocyte from litter mate_3

data_processing:
Quality trimming using FastQC and Fastx-toolkit
Alignment using STAR v2.5.3a. to mm10
Differential expression analysis using DESeq2
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include expected count, FPKM, and TPM values for each sample

extract_protocol:
Total RNA of 1 mg was extracted from each sample
The mRNA library (insert size of ~300 bp) was created using the TruSeq RNA Library Preparation kit v2 (Illumina)

value: FVB/NJ

value: Lymph node

value: 20 months

value: wildtype


title: CD4+lymphocyte from litter mate_2

description: CD4+lymphocyte from litter mate_2

value: 18 months


title: cd8_cd5hi_3


title: CD4+lymphocyte from RHOA transgenic mouse_1

value: 23 months

value: RHOA G17V


title: 10d_WT-179_RNA-seq

city: Nanjing

first: Mei

address: Nanjing Medical University, 101 Longmian Ave., Nanjing, Jiangsu

institute: Nanjing Medical University

data_processing:
Illumina Casava1.8 software used for basecalling
For RNA-seq and CLIP-seq, clean reads were mapped to the mouse genome (mm10) using TopHat (version 2.0.9) with a GTF file download from Ensemble database with maximum 2-base mismatch. Reads from small RNA-seq were mapped to miRBase (version 21.0) by CLC genomics_workbench (version 5.5) without base mismatch
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include reads numbers, RPKM values for each Sample.

source_name: testis

extract_protocol:
For RNA-seq , RNA was harvested using Trizol reagent and RNA libraries were prepared for sequencing using standard Illumina protocols. For LIN28A-CLIP seq, target RNA fragments were amplificated by ligation of adaptors which was in favour of the target RNA reverse transcription and PCR.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: wild type

value: testis

value: post natal day 10


title: 10d_WT-192_RNA-seq


title: sample_LIN28A_CLIP_RL5i1

data_processing:
Library strategy: CLIP-Seq
Illumina Casava1.8 software used for basecalling
For RNA-seq and CLIP-seq, clean reads were mapped to the mouse genome (mm10) using TopHat (version 2.0.9) with a GTF file download from Ensemble database with maximum 2-base mismatch. Reads from small RNA-seq were mapped to miRBase (version 21.0) by CLC genomics_workbench (version 5.5) without base mismatch
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include reads numbers, RPKM values for each Sample.


title: sample_LIN28A_CLIP_RL5i3


title: 10d_cKO-201_RNA-seq

value: Lin28a knockout


title: CD4+lymphocyte from RHOA transgenic mouse_2


title: 10d_WT-155_RNA-seq


title: sample_LIN28A_CLIP_RL5i7


title: cd8_cd5lo_1


title: Th2-cult-OTII-KO2

city: Rotterdam

first: Ralph

last: Stadhouders

address: Dr. Molewaterplein 50

department: Pulmonary Medicine

country: Netherlands

institute: Erasmus Medical Center

data_processing:
Reads were aligned using HISAT2 v2.0.4. (RNA-seq) with default parameters.
Sorted and indexed bam files were parsed to HOMER to creat tag directories without duplicates and bedgraphs for browser visualization
RPKM generation, sample scaling and statistical analysis were performed using HOMER and its implementation of the R package DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: RPKM expression values

source_name: Spleen

growth_protocol: Naive T cells from spleen were differentiated towards a Th2 phenotype by culturing them on anti-CD3/CD28 coated plates in IMDM medium for 7 days in the presence of IL‐4, anti‐IFN‐γ and anti‐IL‐12/23.

extract_protocol:
Naive T cells were obtained from spleens of OTII-transgenic mice. Th2 cells were obtained from Gata3-YFP reporter ('GATIR') or OTII-transgenic mice suffering from an allergen-driven airway inflammation. Cells were FACS-sorted using specific antibodies and YFP as described in detail in the manuscript.
RNA-Seq: Smart-seq2 library construction (as described in Picelli et al. Nature Methods (2013))

value: FACS-purified lymphocytes


title: Th2-cult-OTII-KO1


title: Th2-cult-OTII-WT1

value: OTII


title: 10d_cKO-182_RNA-seq


title: Th2-cult-OTII-WT2


title: Th2-MLN-OTII-KO2

source_name: mediastinal lymph node (MedLN)


title: Th2-MLN-OTII-KO3


title: Th2-MLN-OTII-WT3


title: Th2-MLN-OTII-KO4


title: Th2-MLN-OTII-KO1


title: Th2-MLN-OTII-WT2


title: Th2-MLN-GATIR-WT3

value: GATIR


title: Th2-MLN-GATIR-WT2


title: CD4+lymphocyte from litter mate_1

description: CD4+lymphocyte from litter mate_1


title: Th2-MLN-OTII-WT1


title: Th2-MLN-GATIR-WT1


title: Th2-BAL-GATIR-WT3

source_name: bronchoalveolar lavage (BAL)


title: Th2-BAL-GATIR-WT2


title: 10d_cKO-230_RNA-seq


title: CD4+lymphocyte from RHOA transgenic mouse_3


title: Enlarged lymph node  from RHOA transgenic mouse_3

source_name: Lymph node


title: Lymph node from litter mate_3

description: Lymph node from litter mate_3


title: iAD mouse negative Dox

city: Baltimore

first: Yohan

last: Suryo Rahmanto

address: CRB-2, Rm 376, 1550 Orleans St

department: Department of Pathology

institute: Johns Hopkins School of Medicine

data_processing:
Bioinformatics analysis was performed using Galaxy, an open access web-based program that contains a variety of next-generation sequencing analysis tools. Reads were processed and aligned to Mus musculus reference genome build mm10 using TopHat gapped-read mapper (ver. 2.1.0). The aligned reads were processed with Cufflinks transcript assembly (ver. 2.2.1.0), and output of the resulting GTF files was amalgamated to UCSC hg19 or mm10 RefSeq genes annotation files using Cuffmerge (ver. 2.2.1.0). Differential expression analysis was performed using Cuffdiff (ver. 2.2.1.3) to produce a list of genes whose expression changes were significant (FDR < 0.05) between the groups tested.
Genome_build: mm10

source_name: uterine tumor

growth_protocol: Generation of Arid1aflox/flox mice on the C57BL/6 background was previously described (PMID: 24899687). Cre-mediated deletion of Arid1a removes exon 8 and introduces a frameshift mutation and a premature stop codon (p.Gly809Hisfs*6; wild type ARID1A protein has 2283 amino acids). Ptenflox/flox mice on the BALB/c background (Strain C;129S4-Ptentm1Hwu/J) were obtained from the Jackson Laboratory . Cre-mediated deletion of Pten removes exon 5 and introduces a frameshift mutation (p.Val85Glyfs*14). Arid1aflox/flox;Ptenflox/flox mice were generated by intercrossing these transgenic strains. In order to express Cre recombinase specifically in the mouse uterine epithelium for all subsequent genetic alterations described, we used Pax8-Cre mice which were generated by crossing mice expressing the reverse tetracycline-controlled transactivator (rtTA) under the control of the Pax8 promoter (Pax8-rtTA) with mice expressing Cre recombinase in a tetracycline-dependent manner (TetO-Cre) (PMID: 24332043).  To generate mouse models with Arid1a and Pten individual or combined knockout in uterine epithelium, we crossed Pax8-Cre mice with Arid1aflox/flox mice, Ptenflox/flox mice, and Arid1aflox/flox;Ptenflox/flox mice. All experimental mice were maintained on a mixed genetic background (C57BL/6, BALB/c, and S129). The resultant genotype of each model was confirmed by genomic DNA PCR, using primer sequences described previously (PMID: 24899687). The wild-type, floxed, and recombined Arid1a generate 669, 845, and 298 bp PCR products, respectively. The predicted PCR products for wild-type, flox, and recombined Pten are 1018, 1100, and 400 bp, respectively. Knockout was initiated by treating mice with doxycycline either through oral gavage (2 mg/mouse/day) or subcutaneous implantation of doxycycline pellets (200 mg) when they reached puberty (6-8 weeks old). Hysterectomy (either unilateral or bilateral) was performed 2 weeks, 6 weeks, 10 weeks, 12 weeks, or 16 weeks later. All of the animal procedures were approved by the Johns Hopkins University Animal Care Committee.

extract_protocol:
Total RNA was isolated from iPAD and iPD mouse uterine tissues using the Qiagen RNeasy Plus Mini Kit. Uteri from iAD mice were harvested and subjected to epithelial cells isolation using Multi Tissue Dissociation Kit 1, CD45 MicroBeads, and CD326 MicroBeads (all from Miltenyi Biotech) according to manufacturer protocols. Total RNA from the isolated iAD mice uterine epithelial cells were extracted using the Qiagen RNeasy Plus Micro Kit.
RNA quality was assessed using the Agilent 2100 Bioanalyzer RNA Nano Chip. RIN values ranged from 5.8 to 7.8 for iPD and iPAD mice uteri, and 7.0 to 9.4 for iAD mice uterine epithelial cells. RNA-sequencing was performed by GeneWiz, Inc. on the Illumina HiSeq2500 platform, in a 2x150 bp paired-end high output V4 chemistry configuration.

treatment_protocol: Uteri from 4 iPAD and 4 iPD mice were harvested two weeks after gene knockout (by feeding doxycycline). Hematoxylin-and-eosin stained tissue sections were prepared, and the diagnosis of endometrioid carcinoma from all iPAD mice and EIN from all iPD mice was confirmed by histopathology. Uteri from iAD mice were harvested with and without two weeks of feeding doxycycline.

value: uterine tumor

value: iAD (Arid1aflox/flox)

value: No treatment

tag: mouse id


title: Lymph node from litter mate_2

description: Lymph node from litter mate_2

value: FVB/NJ


title: iAD mouse positive Dox

value: Doxycycline oral gavage (2 weeks)


title: Enlarged lymph node  from RHOA transgenic mouse_2

value: Lymph node

value: 20 months


title: Lymph node from RHOA transgenic mouse_1

description: Lymph node from RHOA transgenic mouse_1

value: 21 months


title: Enlarged lymph node  from RHOA transgenic mouse_4


title: iPD mouse4

description: iPAD_versus_iPD.tabular, P86

data_processing:
Bioinformatics analysis was performed using Galaxy, an open access web-based program that contains variety of next-generation sequencing analysis tools. Reads were processed and aligned to Mus musculus reference genome build mm10 using TopHat gapped-read mapper (ver. 2.1.0). The aligned reads were processed with Cufflinks transcript assembly (ver. 2.2.1.0), and output of the resulting GTF files was amalgamated to UCSC mm10 RefSeq genes annotation files using Cuffmerge (ver. 2.2.1.0). Differential expression analysis was performed using Cuffdiff (ver. 2.2.1.3) to produce a list of genes whose expression changes were significant (FDR < 0.05) between the groups tested.
Genome_build: mm10
Supplementary_files_format_and_content: Cuffdiff file

extract_protocol:
Generation of Arid1aflox/flox mice on the C57BL/6 background was previously described (Gao et al., 2008). Cre-mediated deletion of Arid1a removed exon 8 and introduced a frameshift mutation and a premature stop codon (p.Gly809Hisfs*6; wild type ARID1A protein has 2283 amino acids). Ptenflox/flox mice on the BALB/c background (Strain C;129S4-Ptentm1Hwu/J) were obtained from the Jackson Laboratory (Groszer et al., 2001). Cre-mediated deletion of Pten removed exon 5 and introduced a frameshift mutation (p.Val85Glyfs*14). Arid1aflox/flox;Ptenflox/flox mice were generated by intercrossing these transgenic strains. In order to express Cre recombinase specifically in the mouse uterine epithelium for all subsequent genetic alterations described, we used Pax8-Cre mice which were generated by crossing mice expressing the reverse tetracycline-controlled transactivator (rtTA) under the control of the Pax8 promoter (Pax8-rtTA) with mice expressing Cre recombinase in a tetracycline-dependent manner (TetO-Cre) (Perets et al., 2013).  To generate mouse models with Arid1a and Pten individual or combined knockout in uterine epithelium, we crossed Pax8-Cre mice with Arid1aflox/flox mice, Ptenflox/flox mice, and Arid1aflox/flox;Ptenflox/flox mice. Figure S4A summarizes the generation of these genetically engineered mice in which Arid1a and Pten can be conditionally knocked out. All experimental mice were maintained on a mixed genetic background (C57BL/6, BALB/c, and S129). The resultant genotype of each model was confirmed by genomic DNA PCR, using primer sequences described previously (Guan et al., 2014). The wild-type, floxed, and recombined Arid1a generate 669, 845, and 298 bp PCR products, respectively. The predicted PCR products for wild-type, flox, and recombined Pten are 1018, 1100, and 400 bp, respectively. Knockout was initiated by treating mice with doxycycline either through oral gavage (2 mg/mouse/day). All of the animal procedures were approved by the Johns Hopkins University Animal Care Committee.
Uteri from 4 iPAD and 4 iPD mice were harvested two weeks after gene knockout (by feeding doxycycline). Hematoxylin-and-eosin stained tissue sections were prepared, and the diagnosis of endometrioid carcinoma from all iPAD mice and EIN from all iPD mice was confirmed by histopathology. Total RNA was isolated from mouse uterine tissues using the Qiagen RNeasy Plus Mini Kit. RNA quality was assessed using the Agilent 2100 Bioanalyzer RNA Nano Chip. RIN values ranged from 5.8 to 7.8. RNA-sequencing was performed by GeneWiz, Inc. on the Illumina HiSeq2500 platform, in a 2x100 bp paired-end high output V4 chemistry configuration.

value: iPD (Ptenflox/flox)


title: Enlarged lymph node  from RHOA transgenic mouse_1

value: RHOA G17V


title: Th2-BAL-GATIR-WT1

tag: genotype/variation


title: Lymph node from litter mate_1

description: Lymph node from litter mate_1

tag: age

value: wildtype


title: iPD mouse3

description: iPAD_versus_iPD.tabular, P62


title: iPD mouse2

description: iPAD_versus_iPD.tabular, P61


title: Lymph node from RHOA transgenic mouse_2

description: Lymph node from RHOA transgenic mouse_2


title: iPAD mouse3

description: iPAD_versus_iPD.tabular, AP22

value: iPAD (Arid1aflox/flox and Ptenflox/flox)


title: iPAD mouse1

description: iPAD_versus_iPD.tabular, 2AP17


title: iPAD mouse2

description: iPAD_versus_iPD.tabular, AP18


title: iPAD mouse4

description: iPAD_versus_iPD.tabular, AP30


title: delta ICP27  Rep. 2

city: München

first: Caroline

last: Friedel

address: Amalienstr. 17

department: Institut für Informatik

country: Germany

institute: Ludwig-Maximilians-Universität München

description:
Purified from total RNA using 4sU-tagging
gene_counts.tsv

data_processing:
Reads were mapped against the human genome (GRCh37/hg19), HSV-1 genome (Human herpesvirus 1 strain 17, JN555585, terminal repeats masked) and rRNA sequences using ContextMap v2.7.9
Number of read fragments per gene were determined using featureCounts and gene annotations from Ensembl (version 87 for GRCh37). HSV-1 gene annotations were obtained from GenBank (GenBank accession code: JN555585).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Gene counts

growth_protocol: Human foreskin fibroblasts were cultured in DMEM supplements with 10% FBS and P/S. Cells at passage 13 were infected at 60% confluency 24h after the last split.

extract_protocol:
Total RNA was prepared using Trizol. Newly transcribed RNA was purified as described in Rädle et al., JoVE 2013.
TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (only for total RNA). No rRNA depletion was performed for the 4sU-RNA samples!

treatment_protocol: Cells were infected for 10 min with the indicated viruses at an MOI of 10 followed by PBS wash and media replacement. 500µM 4-thiouridine was added to the cell culture at the indicated time points.

value: human foreskin fibroblasts

tag: time post infection

value: 60 min 500 u 4sU at 7-8hpi

tag: infection with

value: delta ICP27


title: WT strain 17 Rep. 2

tag: cell type

value: WT strain 17


title: delta vhs  Rep. 2

value: delta vhs


title: delta ICP22 Rep. 2

value: 60 min 500 u 4sU at 11-12hpi

value: delta ICP22


title: TsK  Rep. 2

value: TsK, 39°


title: delta ICP4  Rep. 1

value: delta ICP4


title: delta ICP22  Rep. 1


title: delta IPC0 Rep. 2

value: delta IPC0


title: delta ICP27  Rep. 1


title: delta IPC0  Rep. 1


title: Mock Rep. 1

value: 60 min 500 u 4sU prior to infection

value: Mock (1h 4sU)


title: T47D_sh97_WT_RING1B_rescue_24hr_rep1

city: Miami

first: Lluis

last: Morey

department: Sylvester Comprehensive Cancer Center

institute: University of Miami Miller School of Medicine

data_processing:
ATAC-seq: Paired-end ATAC-seq fastq files were processed using the ATAC-seq Pipeline from the Kundaje Lab (https://github.com/kundajelab/atac_dnase_pipelines). Reads were trimmed using cutadapt v1.9.1 using the following parameters: -m 5. Reads were aligned to the hg19 genome using Bowtie2 v2.2.6, and the SAMtools v1.7 was used to convert the output files into the BAM format using default parameters. Duplicate reads were removed using Picard tool v1.126 using default parameters. Shuf was used to subsample reads to 50 million. Peaks were called using MACS2 v2.1.0 using default parameters
ChIP-seq: Single-end ChIP-seq fastq files were processed using the AQUAS ChIP-seq Pipeline from the Kundaje Lab (https://github.com/kundajelab/chipseq_pipeline). Reads were trimmed using cutadapt v1.9.1. Reads were aligned to the hg19 genome using Bowtie2 v2.2.6, and the SAMtools v1.7 was used to convert the output file to the BAM format. Duplicates were removed using Picard Tools v1.126. Peaks were called/signal tracks were generated using MACS2 v2.1.0, and peaks with fold change > 4 compared to input and a FDR < 0.05 were used for subsequent analysis. Only default parameters were used.
RNA-seq: Single-end RNA-seq fastq files were processed using the LSF-RNAseq Pipeline from Diderote (https://github.com/diderote/LSF-RNAseq). Reads were trimmed using cutadapt v2.3 with the following parameters: -j 4 --netseq-trim=20 -m 18. Reads were aligned to the hg19 genome using STAR v2.7.1a along default parameters, and RSEM v1.3.0 was used to obtain the expected gene counts against the human refseq also along default parameters.
RNA-seq differential expression was determined using DESeq2 v1.18.1 and R (version 3.4.1) with q-value <0.05. Ruvseq v1.12.0 was used for factor analysis to remove count based on ERCC spike-in. GSEA analysis was performed using gsea-3.0.jar. These additional analysis steps were included in the Diderote LSF-RNAseq Pipeline.
Homer annotatePeaks v4.8.3 was used for peak  annotation and for assigning peaks to genes.
Bedtools v2.26.0 intersect was used to determine peak overlaps.
Genome_build: hg19
Supplementary_files_format_and_content: The processed data files are in bigWig format containing the indexed tag chromosome coordinates and score produced from MACS2 peak calling.

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-WT-RING1B plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 15% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The re-sorted cells were expanded, and the exognous WT-RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before collection and RNA extraction for library preparation.

extract_protocol:
RNA was isolated from frozen cell pellets using the TRIzol reagent (Thermo Fisher Scientific cat# 15596018) according to manufacturer's instructions. RNA quantity was measured on a nanodrop, and RNA was diluted to 100 ng/ul or less. A total of 1.5 ug to 2 ug of RNA in 20 ul of nuclease-free water was treated using the TURBO DNA-free Kit (Thermo Fisher AM1907) along the Routine DNase treatment guideline.
Library preparation was performed on Dnase-treated RNA using the Illumina Truseq Stranded Total RNA Library Kit (Illumina 20020596). The Truseq RNA UD Indexes were used for the adapter ligation step (Illumina 20020591). Samples were normalized to a concentration of 250 ng for input, and 1 ul of ERCC spike-in Mix 1 (Thermo Fisher 4456740) diluted 1:200 was added to the samples. Minor protocol modifications were made: 1) during the "Elution 2 - Frag - Prime" step, samples were incubated for 6 minutes at 94°C instead of the listed 8 minutes, and 2) 10 cycles were used in the amplification step under the "Enrich RNA Fragments" section instead of the suggested 15 cycles.

value: Mammary gland ductal carcarcinoma epithelial cells derived from metastatic site

tag: cell source

value: ATCC #HTB-133

tag: modifications


title: WT strain 17 Rep. 1


title: line #3 iron treatment

city: Stanford

first: Lei

last: Tian

state: CA

address: 1215 Welch Rd

institute: Stanford's Postdoctoral Scholar programs

description: C2 FE (2-CTR)

data_processing:
TrimGalore
STAR
featureCounts
Genome_build: hg38
Supplementary_files_format_and_content: Number of uniquely mapped RNA-seq counts located in each gene

source_name: iPSC-CM

growth_protocol: Cardiogenic differentiation of human iPSC was performed using a chemically defined protocol using activator and inhibitors of the Wnt signaling pathway.

extract_protocol:
Total RNA was isolated using micro RNeasy kit (QIAGEN) in accordance to manufacturer’s instructions, followed by DNAse treatment.
RNA-seq library was constructed from 100 ng of the total RNA using the KAPA stranded RNA-seq kit with RiboErase following manufacturer's instructions

treatment_protocol: iPSC-CMs were treated with divalent iron (100 μM) or ascorbic acid as a control for a period of 5 days.

tag: treatment

value: iron treatment

value: iPSC-CM

tag: cell origin

value: Human


title: line #2 vehicle treatment (ascorbic acid)

description: C3 AA (15-CTR)

value: Control


title: line #1 iron treatment

description: C1 FE (273-CTR)


title: line #3 vehicle treatment (ascorbic acid)

description: C2 AA (2-CTR)


title: line #1 vehicle treatment (ascorbic acid)

description: C1 AA (273-CTR)


title: T47D_sh97_WT_RING1B_rescue_0hr_rep1

tag: cell line


title: T47D_sh97_WT_RING1B_rescue_0hr_rep2


title: T47D_sh97_WT_RING1B_rescue_24hr_rep2


title: line #2 iron treatment

description: C3 FE (15-CTR)


title: iPD mouse1

description: iPAD_versus_iPD.tabular, P60

value: iPD (Ptenflox/flox)

value: Doxycycline oral gavage (2 weeks)


title: Mock Rep. 2

value: human foreskin fibroblasts


title: T47D_E2_RING1B_ChIPexo

data_processing:
ATAC-seq: Paired-end ATAC-seq fastq files were processed using the ATAC-seq Pipeline from the Kundaje Lab (https://github.com/kundajelab/atac_dnase_pipelines). Reads were trimmed using cutadapt v1.9.1 using the following parameters: -m 5. Reads were aligned to the hg19 genome using Bowtie2 v2.2.6, and the SAMtools v1.7 was used to convert the output files into the BAM format using default parameters. Duplicate reads were removed using Picard tool v1.126 using default parameters. Shuf was used to subsample reads to 50 million. Peaks were called using MACS2 v2.1.0 using default parameters
ChIP-seq: Single-end ChIP-seq fastq files were processed using the AQUAS ChIP-seq Pipeline from the Kundaje Lab (https://github.com/kundajelab/chipseq_pipeline). Reads were trimmed using cutadapt v1.9.1. Reads were aligned to the hg19 genome using Bowtie2 v2.2.6, and the SAMtools v1.7 was used to convert the output file to the BAM format. Duplicates were removed using Picard Tools v1.126. Peaks were called/signal tracks were generated using MACS2 v2.1.0, and peaks with fold change > 4 compared to input and a FDR < 0.05 were used for subsequent analysis. Only default parameters were used.
RNA-seq: Single-end RNA-seq fastq files were processed using the LSF-RNAseq Pipeline from Diderote (https://github.com/diderote/LSF-RNAseq). Reads were trimmed using cutadapt v2.3 with the following parameters: -j 4 --netseq-trim=20 -m 18. Reads were aligned to the hg19 genome using STAR v2.7.1a along default parameters, and RSEM v1.3.0 was used to obtain the expected gene counts against the human refseq also along default parameters.
RNA-seq differential expression was determined using DESeq2 v1.18.1 and R (version 3.4.1) with q-value <0.05. Ruvseq v1.12.0 was used for factor analysis to remove count based on ERCC spike-in. GSEA analysis was performed using gsea-3.0.jar. These additional analysis steps were included in the Diderote LSF-RNAseq Pipeline.
Homer annotatePeaks v4.8.3 was used for peak  annotation and for assigning peaks to genes.
Bedtools v2.26.0 intersect was used to determine peak overlaps.
ChIP-exo: FASTQ data were processed using cutadapt v1.15 (--netseq-trim=20 -m 10) to remove low quality reads and then aligned to the human genome hg19 using BWA v0.7.13 (aln -q 5 -l 32 -k 2). Peaks and motif subtypes were determined using ChExMix v0.41 and MEME v5.0.5 after filtering blacklisted regions and enabling a probability-based duplicate filter (--readfilter). Motif matching and motif distributions were determined using MEME-chip v5.0.2 with the JASPAR 2016 motif database. Peak distributions were determined using gaussian kernel function estimates of ChExMix motif aligned peaks.
Genome_build: hg19
Supplementary_files_format_and_content: The processed data files are in bigWig format containing the indexed tag chromosome coordinates and score produced from MACS2 peak calling.

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS, 10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. Cells were cultured in hormone-deprived media for 3 days. After hormone deprivation, cells were treated with 10 nm of E2 for 45 minutes before crosslinking with formaldehyde and collection for ChIP-exo.

extract_protocol:
The ChIP-exo 5.0 experiment was performed using the published protocol (Rossi et al. Nat Comm 2018) with minor adaptations in regards to chromatin preparation and the IP step. Cells were grown to 80% confluence on 150cm plates and washed once with PBS and fixed with 1% formaldehyde (Thermo Fisher #28908) in 10 ml PBS for 10 min. To quench the cross-linking reaction, 1/20 volume of 2.5 M glycine was added to the plate for a final concentration of 0.125M for 5 min. The supernatant was aspirated, and the cells were washed twice with 5 ml PBS and harvested on ice using cell scrapers into 15ml sonication tubes (Diagenode cat# C01020031). Cells were pelleted at 1000g for 5 min at 4°C, washed 1x with 5 ml cold PBS, and pelleted once more. Cell pellet collected from 6 plates of cells was resuspended in 3 ml of Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.5; 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100), allowed to rotate for 10 min at 4°C, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 2 (10 mM Tris-HCL pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), allowed to rotate for 10 min at room temperature, and pelleted at 1000 x g for 5 min at 4°C. Cells were then resuspended in 3 ml of Lysis Buffer 3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and then sonicated on a Bioruptor Pico at 4°C for 20min of 30’’ ON-OFF cycles with 1.2 g of sonication beads. After sonication, samples were transferred to 2 separate 1.5 ml microtube and centrifuged at 20, 000 x g for 15 minutes at 4°C. The supernatant was transferred to new 15 ml tubes. To check sonication efficiency, 20µl of sonicated samples was transferred to a new microtube with 80µl PBS and incubated at 65°C for 3hr on an Eppendorf Thermomixer shaking at 1000rpm to decrosslink. DNA was purified with the QIAquick PCR Purification Kit (Qiagen cat# 28106) and eluted in 50µl EB buffer. 1 ug of decrosslinked chromatin from each sample were run in a 1% agarose gel at 100V for 45min and imaged on a Bio-rad ChemiDoc XRS+. DNA concentration in the sonicated decrosslinked samples were measured via qubit, and this concentration was then used to estimate the concentration of chromatin in the un-decrosslinked samples. 120 ug of total chromatin were transferred equally to two 1.5ml LoBind tubes (Eppendorf cat# 0030108051) and each was brought up to a final volume of 500µl with Lysis Buffer 3. 10 ug of antibodies were use for transcription factor ChIP (see Key Resources table for list of antibodies), which was divided equally into two and individually pre-bound to 10 ul of Protein A Mag Sepharose Beads (GE Healthcare 28944006). The beads were washed in 1 ml of 0.5% BSA in 1x PBS three times before and after overnight pre-binding at 4°C. The chromatin samples were added to the pre-bound Dynabeads and rotated end-to-end overnight at 4°C. The beads were collected on the magnetic rack and sequentially washed with 500 uL FA Lysis Buffer, NaCl Buffer, LiCl Buffer, and 10 mM Tris-HCl buffer pH 8.0, all at 4°C. The rest of the reaction and the library preparation were performed exactly as described in the previously cited paper.
Library preparation was performed as described (Rossi et al. nat Comm 2018) with minor adaptations in regards to the oligonucleotides used. NEBNext Multiplex Oligos for Illumina Set 1 was used in place of ExA2_iNN. NEBNext Universal PCR Primer for Illumina was used instead of ExA1-58. All other oligos were synthesized from Sigma except for ExA1-SSL_N5, which was ordered from IDT with 5 random basepairs at the beginning of the oligo.

value: stimulation with E2

tag: chip antibody

value: Active Motif C39663


title: T47D_HD_RING1B_ChIPexo

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS, 10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. Cells were cultured in hormone-deprived media for 3 days. After hormone deprivation, cells were crosslinked with formaldehyde and collected for ChIP-seq.

value: hormone-depleted (HD) condition


title: T47D_E2_GRHL2_ChIPexo

value: Sigma Aldrich HPA004820


title: T47D_HD_GRHL2_ChIPexo


title: delta vhs  Rep. 1

value: 60 min 500 u 4sU at 7-8hpi

value: delta vhs


title: T47D_E2_ERa_ChIPexo

value: Diagenode C15410196


title: T47D_E2_FOXA1_ChIPexo

value: Abcam ab23738


title: TsK  Rep. 1

value: TsK, 39°


title: T47D_sh97_12hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shRING1B cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 12 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.

extract_protocol:
RNA was isolated from frozen cell pellets using the TRIzol reagent (Thermo Fisher Scientific cat# 15596018) according to manufacturer's instructions. RNA quantity was measured on a nanodrop, and RNA was diluted to 100 ng/ul or less. A total of 1.5 ug to 2 ug of RNA in 20 ul of nuclease-free water was treated using the TURBO DNA-free Kit (Thermo Fisher AM1907) along the Routine DNase treatment guideline.
Library preparation was performed on Dnase-treated RNA using the Illumina Truseq Stranded Total RNA Library Kit (Illumina 20020596). The Truseq RNA UD Indexes were used for the adapter ligation step (Illumina 20020591). Samples were normalized to a concentration of 500 ng for input, and 1 ul of ERCC spike-in Mix 1 (Thermo Fisher 4456740) diluted 1:200 was added to the samples. Minor protocol modifications were made: 1) during the "Elution 2 - Frag - Prime" step, samples were incubated for 6 minutes at 94°C instead of the listed 8 minutes, and 2) 10 cycles were used in the amplification step under the "Enrich RNA Fragments" section instead of the suggested 15 cycles.

value: Mammary gland ductal carcarcinoma epithelial cells derived from metastatic site

value: ATCC #HTB-133

value: pLKO-shRING1B stable


title: T47D_sh97_24hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shRING1B, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 24 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_HD_FOXA1_ChIPexo

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS, 10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. Cells were cultured in hormone-deprived media for 3 days. After hormone deprivation, cells were crosslinked with formaldehyde and collected for ChIP-exo.


title: T47D_HD_ERa_ChIPexo


title: T47D_sh97_8hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shRING1B, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 8 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_shCtrl_12hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shCtrl, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 12 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.

value: pLKO-shCtrl stable


title: T47D_shCtrl_24hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shCtrl, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 24 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_shCtrl_4hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shCtrl, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 4 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_shCtrl_0hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shCtrl, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days and collected. Pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_shCtrl_8hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shCtrl, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 8 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_sh97_4hr_RNAseq_rep1

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shRING1B, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days before 4 hours of 10 nM E2 treatment. Cells were then collected, and the pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_sh97_24hr_RNAseq_rep1


title: T47D_sh97_8hr_RNAseq_rep1


title: T47D_sh97_12hr_RNAseq_rep1


title: T47D_shCtrl_24hr_RNAseq_rep1


title: T47D_shCtrl_8hr_RNAseq_rep1


title: T47D_sh97_0hr_RNAseq_rep1

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with pLKO-shRING1B, cells were selected and maintained in media containing 2 ug/ml puromycin. Cells were then hormone deprived for 3 days and collected. Pellets were frozen at -80°C for RNA extraction at a later date.


title: T47D_shCtrl_12hr_RNAseq_rep1


title: T47D_RING1B_I53A_24hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-RNF2-I98A plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 25% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The re-sorted cells were expanded, and the mutant RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before 24 hours of 10 nM E2 treatment. Cells were then collected, and RNA was extracted for library preparation.


title: T47D_shCtrl_0hr_RNAseq_rep1


title: T47D_shCtrl_4hr_RNAseq_rep1


title: T47D_RING1B_I53A_0hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-RNF2-I53A plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 25% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The re-sorted cells were expanded, and the mutant RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before collection and RNA extraction for library preparation.


title: T47D_sh97_4hr_RNAseq_rep2


title: T47D_RING1B_R98A_24hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-RNF2-R98A plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 25% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The re-sorted cells were expanded, and the mutant RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before 24 hours of 10 nM E2 treatment. Cells were then collected, and RNA was extracted for library preparation.


title: 19_DOX, primary cell tubuloids

city: Saint Louis

first: Eryn

middle: E

last: Dixon

state: MO

address: 600 S Euclid Ave

department: Renal Division/Internal Medicine

institute: Washington University in St. Louis

data_processing:
For Expression Counts using BasePair, trim smallRNA adapters, align reads using STAR, count using FeatureCounts
For Differential Expression via DESeq2 algorithm, generate up/down lists, gct/cls files, volcano plot, heatmap
Genome_build: STAR alignment using NCBI reference (mouse genome mm10 or GRCm38)
Supplementary_files_format_and_content: Expression count

source_name: primary cell tubuloids

growth_protocol: Primary cell fragments were plated in Matrigel, differentiated with growth factors, and cultured for seven days before extraction.

extract_protocol:
Tubuloids were removed from Matrigel using Corning recovery solution and RNA was isolated with Rneasy Plus Mini Kit.
Libraries were made with Clontech SMART-Seq v4 Ultra Low Input RNA kit (minimum concentration 2 ng/uL; with poly-A selection).

treatment_protocol: Tubuloids were treated for three days with either DMSO or DOX on the third day of culture, following differentiation.

value: postnatal day 7-10 kidney

tag: genotype

value: Pkd2fl/fl Pax8rtTA TetOCre mTmG

value: DOX


title: 12_DOX, primary cell tubuloids


title: 21_DOX, primary cell tubuloids


title: T47D_sh97_0hr_RNAseq_rep2


title: T47D_RING1B_WT_24hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. Cells were hormone-deprived in charcoal-stripped media for 3 days before 24 hours of treatment with 10 nM of E2. Cells were then collected, and RNA was extracted for library preparation.

value: Parental


title: 5_DMSO, primary cell tubuloids

value: DMSO


title: 3_DMSO, primary cell tubuloids


title: T47D_RING1B_WT_0hr_RNAseq_rep2

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. Cells were hormone-deprived in charcoal-stripped media for 3 days before collection and RNA extraction for RNA-seq library preparation.


title: 9_DOX, primary cell tubuloids


title: 20_DMSO, primary cell tubuloids


title: T47D_RING1B_I53A_24hr_RNAseq_rep1

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-RNF2-I98A plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 25% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The resorted cells were expanded, and the mutant RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before 24 hours of 10 nM E2 treatment. Cells were then collected, and RNA was extracted for library preparation.


title: 15_DMSO, primary cell tubuloids


title: Ctl3, primary cell tubuloids

treatment_protocol: Tubuloids were plated and then received either growth factor enriched media with or without GDNF for a 24 hour pulse. After this timepoint, GDNF was removed from pulsed cultures and then all tubuloids continued to receive growth factor enriched media until extraction.

value: control


title: Ctl14, primary cell tubuloids


title: Ctl7, primary cell tubuloids


title: Ctl21, primary cell tubuloids


title: T47D_RING1B_WT_24hr_RNAseq_rep1


title: T47D_RING1B_I53A_0hr_RNAseq_rep1


title: T47D_RING1B_WT_0hr_RNAseq_rep1


title: T47D_RING1B_R98A_0hr_RNAseq_rep1

growth_protocol: T47D were maintained at 37°C with 5% CO2 and passaged every 2-3 days at a 1:3 split ratio, according to ATCC recommendations. Complete culture media comprised of RPMI-1640 that was supplemented with 10% FBS,10μg/ml insulin (Sigma-Aldrich cat# 91077C), 1x penicillin/streptomycin, and 1x glutamax. After transduction with lentivirus made using the pLVK-RNF2-R98A plasmid, Fluorescence-Activated Cell Sorting (FACS) was used to select cells whose mCherry expression levels were in the top 25% of all cells. Cells were expanded, and FACS was performed once more to remove any cells without mCherry expression. The re-sorted cells were expanded, and the mutant RING1B expression level was compared with the endogenous RING1B expression level using immunoblot to make sure that the two levels are similar. Cells were then transduced with pLKO-shRING1B, and transduced cells were selected using a puromycin concentration of 2ug/ml. After 7 days of selection, 5 x 10^5 cells were plated into each well on a six-well plate in charcoal-stripped media containin 2ug/ml puromycin. Cells were hormone-deprived for 3 days before collection and RNA extraction for library preparation.