title: inf_BMDMs 37 scRNA-seq

data_processing:
Fastq reads were demultiplexed to single cells based on the CEL-Seq barcode located in positions 7-12 of R1.
R2 reads were filtered to contain the GFP sequence preceding the MIB (positions 1-37 of R2; GFP sequence: TGGGATTACACATGGCATGGATGAACTATACAAATAA, for ∆sseJ the sequence is TGGGATCACACATGGCATGGATGAACTATACAAATAA due to mutation in the plasmid during cloning). To account for sequencing errors, 1 mismatch was allowed for the GFP sequence identification.
The sequence of 10 nucleotides following the GFP (positions 38-47 of R2) is the bacterial barcode, i.e., the MIB sequence. In each single-cell, we counted the number of reads that correspond to each bacterial barcode allowing 1 mismatch in the MIB sequence
Using this procedure, we generated a matrix with the counts of each mutant (MIB) in each cell
Supplementary files format and content: Text files include MIB counts table for each mutant in each cell

extract_protocol:
Cells were harvested 20 hours post infection by a PBS wash followed by incubation of 10 min with ice-cold EDTA (5mM) and analyzed by flow cytometry (BD FACS Aria III) under continuous cooling at 4°C. Infected single cells were sorted into twin.tec® PCR plate 384 LoBind® containing 1.2μl of Cel-seq Lysis buffer per each well, placed immediately on dry ice and stored in minus 80.
Single cells were processed for libraries generation according to the Cel-seq protocol (Hashimshony et al., 2016)

treatment_protocol: LB-grown cultures of WT or each of 24 MIB-tagged SPI-2 mutant strains were pooled into one tube and opsonized with 20% mouse serum for 30 min and washed with PBS. BMDMs were infected with the pool of SPI-2 mutants at MOIs of 2:1 and spun down for 5 min at 400g to synchronize internalization. After 30 min, cells were washed with media containing 50 μg/ml gentamicin to remove S.Tm that were not internalized. Fresh media containing 50 μg/ml gentamicin was then added back to the cells for the duration of infection

tag: time point

value: 20 hours post infection


title: inf_BMDMs 34 scRNA-seq

status: Public on Jun 21 2023

type: SRA

city: Rehovot

first: Noa

last: Bossel Ben-Moshe

address: Hertzl Street

country: Israel

institute: Weizmann

library_source: transcriptomic

molecule: total RNA

organism: synthetic construct

source_name: infected BMDMs

growth_protocol: BMDMs (8x[10^5] cells/well) were plated in non-tissue culture treated six–well plates supplemented with DMEM containing 20% FBS and 25 ng/ml rmM-CSF


title: inf_BMDMs 33 scRNA-seq


title: inf_BMDMs 36 scRNA-seq


title: inf_BMDMs 32 scRNA-seq


title: inf_BMDMs 38 scRNA-seq


title: sifA_inf_bulk

data_processing:
Library strategy: scPAIR-seq
Fastq reads were demultiplexed to samples based on the CEL-Seq barcode located in positions 7-12 of R1.
R2 reads were filtered to contain the GFP sequence preceding the MIB (positions 1-37 of R2; GFP sequence: TGGGATTACACATGGCATGGATGAACTATACAAATAA, for ∆sseJ the sequence is TGGGATCACACATGGCATGGATGAACTATACAAATAA due to mutation in the plasmid during cloning). To account for sequencing errors, 1 mismatch was allowed for the GFP sequence identification.
The sequence of 10 nucleotides following the GFP (positions 38-47 of R2) is the bacterial barcode, i.e., the MIB sequence. In each sample, we counted the number of reads that correspond to each bacterial barcode allowing 1 mismatch in the MIB sequence
Using this procedure, we generated a matrix with the counts of each mutant (MIB) in each sample
Supplementary files format and content: Text files include MIB counts per sample

extract_protocol:
Cells were harvested 4 hours post infection by a PBS wash followed by incubation of 10 min with ice-cold EDTA (5mM) and analyzed by flow cytometry (BD FACS Aria III) under continuous cooling at 4°C. Infected cells were sorted into 1.7ml low–bind tubes containing 25μl of lysis buffer (0.1% Triton in RNase-free water), placed immediately on dry ice and stored in minus 80.
scPAIR-seq relies on the CEL-seq library preparation protocol (Hashimshony et al., 2016) up to in-vitro transcription (IVT) step. After IVT, amplified RNA (aRNA) was treated with EXO-SAP enzyme (15 min at 37°C) to remove unused primers leftovers. aRNA sample was split and half of the sample volume (11 µl) was used to perform nucleic acid purification with RNAClean XP beads. The purified aRNA was converted to cDNA using superscript Ⅲ with specific GFP primer and cDNA was purified with AMPure XP beads. MIB library was amplified by 25 PCR cycles using a nested GFP primer to improve specificity of MIB amplification. To ensure exclusive sequencing of MIB reads by a custom GFP primer in the downstream steps, the nested GFP-primer was attached with a 5′-tail containing Illumina 3′-adaptor that was modified in its binding site for the Illumina sequencing primer

treatment_protocol: LB-grown cultures of WT or each of 24 MIB-tagged SPI-2 mutant strains were opsonized with 20% mouse serum for 30 min and washed with PBS. BMDMs were infected with each of SPI-2 mutants separately at MOIs of 5:1 and spun down for 5 min at 400g to synchronize internalization. After 30 min, cells were washed with media containing 50 μg/ml gentamicin to remove S.Tm that were not internalized. Fresh media containing 50 μg/ml gentamicin was then added back to the cells for the duration of infection

tag: infected with

value: ∆sifA


title: inf_BMDMs 30 scRNA-seq


title: VarsSeqRNA

city: PALO ALTO

first: Rhiju

last: Das

state: CA

address: 279 Campus Dr, B419 Beckman Center

country: USA

institute: Stanford University

data_processing:
UMI-tools was used to extract randomized barcode sequences (one for each transformant) and UMI's (one for each cDNA molecule) from sequencing reads. cutadapt was used remove adapter sequences including indexing primer sequences and the universalcloning linker, additionally trimming bases with Q-score lower than 20.
Sequencing reads were aligned to spliced isoform reference sequences for each intron. For reference sequence annotations, we collated all possible isoforms capturing spliced and unspliced transcripts for each gene (4 isoforms for two-intron RPL7A and 2 isoforms for all other genes), and we generated gff annotation files with gmap. TopHat2 was used to align sequencing reads to these reference isoform annotations, using the following flags: --no-novel-juncs -T --bp-mp 3,1 --b2-rdg 5,1 --b2-rfg 5,1 --segment-length 20 --segment-mismatches 3 --read-gap-length 7 --read-edit-dist 50 -m 1 --max-insertion-length 19 --max-deletion-length 19. We generated fasta files from the resulting alignment files with samtools.
Each aligned sequence was classified into one of three categories: unspliced, spliced at the expected junction, or other. We required an exact agreement with 14 nucleotides representing the unspliced junction or spliced junction. For each barcode, we computed the number of spliced and unspliced reads after deduplicating UMI's. For each barcode, we then computed the retained intron fraction (ratio between unspliced and total read counts) along with the normalized mRNA level (ratio between spliced read count and the transformation frequency from DNA sequencing.)
Assembly: S. cerevisiae intron reference sequences were obtained from Talkish, et al. 2019 (doi: 10.1371/journal.pgen.1008249).Coding ORF annotations were obtained from the Saccharomyces Genome Database for the S288C reference genome. Pre-mRNA reference sequences were obtained using the sacCer3 UCSC genome assembly.
Supplementary files format and content: For each sub-library, in files ending with barcode_umi_spliced.txt we provide read counts for spliced and unspliced RNA for all barcodes and UMI's. Each row has the following format: 12-nucleotide barcode, 10-nucleotide UMI, number of spliced reads, number of unspliced reads, and other reads. In addition, we provide the file designed_var_barcode_spliced.csv, which includes spliced and unspliced read counts per barcode for designed variants after demultiplexing UMI's. Samples are indexed as follows: S1 (QCR9), S2 (RPL28), S3 (RPS9B), S4 (RPL7A), S5 (RPS9A), S7 (RPS14B), and S8 (RPL36B).
Library strategy: Targeted RNA-seq

organism: Saccharomyces cerevisiae

source_name: Library integrated in PLH001

growth_protocol: Intron variant libraries were integrated into the genome of the S. cerevisiae strain PLH001 using homologous recombination and CRISPR/Cas9-induced double stranded breaks to increase efficiency. Our library included seven sub-libraries with variants of the introns in the following genes: QCR9, RPL28, RPS9B, RPL7A, RPS9A, RPS14B, and RPL36B. Integrated constructs included intron variants in their complete gene context with randomized 12N barcodes upstream of introns in 5' UTR regions. For both DNA and RNA sequencing, overnight cultures were grown from sub-library glycerol stocks at 30 oC in YPD liquid medium, diluted to OD600 0.1, and grown to OD600 0.5-0.6.

extract_protocol:
RNA was extracted using the YeaStar RNA Kit (Zymo Research), using 5 μL of Zymolase for 5 mL starting cell culture.
We first depleted the extracted RNA of rRNA using RNase H to deplete rRNA with complementary oligos.We used an RNA Clean and Concentrator-5 (Zymo) column with the size-selection protocol to exclude RNA below a size cutoff of 200 nucleotides. RNA was fragmented with Ambion fragmentation reagents, end-repaired withrSAP (NEB), and then ligated to an adenylated universal DNA cloning linker withT4 RNA ligase 2 truncated KQ (NEB). Excess DNA linker was degraded with 5’ Deadenylase (NEB) and RecJf (NEB). We reverse transcribed RNA with TGIRT enzyme (InGex), using a reverse transcription primer that included a 10N interval for unique molecular identifiers (UMIs). cDNA was amplified using targeted PCR primers for each sub-library, first adding i5/i7 indices along with Illumina adapters in a short indexing PCR. We then carried out an additional PCR reaction with P5/P7 primers to generate the final library and size-selected the library using RNACleanXP beads (Beckman Coulter).

tag: strain

value: Library integrated in PLH001


title: inf_BMDMs 26 scRNA-seq


title: inf_BMDMs 29 scRNA-seq


title: gtgE_inf_bulk

value: ∆gtgE


title: inf_BMDMs 23 scRNA-seq


title: inf_BMDMs 20 scRNA-seq


title: inf_BMDMs 27 scRNA-seq


title: inf_BMDMs 22 scRNA-seq


title: inf_BMDMs 18 scRNA-seq


title: inf_BMDMs 19 scRNA-seq


title: inf_BMDMs 16 scRNA-seq


title: inf_BMDMs 15 scRNA-seq


title: inf_BMDMs 35 scRNA-seq


title: inf_BMDMs 25 scRNA-seq


title: inf_BMDMs 12 scRNA-seq


title: inf_BMDMs 13 scRNA-seq


title: inf_BMDMs 11 scRNA-seq


title: inf_BMDMs 14 scRNA-seq


title: inf_BMDMs 9 scRNA-seq


title: inf_BMDMs 31 scRNA-seq


title: inf_BMDMs 28 scRNA-seq


title: inf_BMDMs 10 scRNA-seq


title: inf_BMDMs 7 scRNA-seq


title: inf_BMDMs 8 scRNA-seq


title: inf_BMDMs 6 scRNA-seq


title: inf_BMDMs 24 scRNA-seq


title: inf_BMDMs 4 scRNA-seq


title: inf_BMDMs 2 scRNA-seq


title: inf_BMDMs 3 scRNA-seq


title: inf_BMDMs 5 scRNA-seq


title: inf_BMDMs 1 scRNA-seq


title: inf_BMDMs 17 scRNA-seq


title: inf_BMDMs 3 scPAIR-seq

data_processing:
Library strategy: scPAIR-seq
Fastq reads were demultiplexed to single cells based on the CEL-Seq barcode located in positions 7-12 of R1.
R2 reads were filtered to contain the GFP sequence preceding the MIB (positions 1-37 of R2; GFP sequence: TGGGATTACACATGGCATGGATGAACTATACAAATAA, for ∆sseJ the sequence is TGGGATCACACATGGCATGGATGAACTATACAAATAA due to mutation in the plasmid during cloning). To account for sequencing errors, 1 mismatch was allowed for the GFP sequence identification.
The sequence of 10 nucleotides following the GFP (positions 38-47 of R2) is the bacterial barcode, i.e., the MIB sequence. We counted the number of reads that correspond to each bacterial barcode allowing 1 mismatch in the MIB sequence
Using this procedure, we generated a matrix with the counts of each mutant (MIB) in the pooled sample
Supplementary files format and content: Text files include MIB counts  for each mutant in each bulk sample

extract_protocol:
Cells were harvested 20 hours post infection by a PBS wash followed by incubation of 10 min with ice-cold EDTA (5mM) and analyzed by flow cytometry (BD FACS Aria III) under continuous cooling at 4°C. Infected cells were sorted into 1.7ml low–bind tubes containing 25μl of lysis buffer (0.1% Triton in RNase-free water), placed immediately on dry ice and stored in minus 80.
scPAIR-seq relies on the CEL-seq library preparation protocol (Hashimshony et al., 2016) up to in-vitro transcription (IVT) step. After IVT, amplified RNA (aRNA) was treated with EXO-SAP enzyme (15 min at 37°C) to remove unused primers leftovers. aRNA sample was split and half of the sample volume (11 µl) was used to perform nucleic acid purification with RNAClean XP beads. The purified aRNA was converted to cDNA using superscript Ⅲ with specific GFP primer and cDNA was purified with AMPure XP beads. MIB library was amplified by 25 PCR cycles using a nested GFP primer to improve specificity of MIB amplification. To ensure exclusive sequencing of MIB reads by a custom GFP primer in the downstream steps, the nested GFP-primer was attached with a 5′-tail containing Illumina 3′-adaptor that was modified in its binding site for the Illumina sequencing primer

tag: replicate


title: inf_BMDMs 21 scRNA-seq


title: inf_BMDMs 2 scPAIR-seq


title: G3 HPK1-M46 rep3 [HWr_HPK1_13049]

city: Houston TX

first: Hua

last: Wang

state: TX

address: 6565 MD Anderson

institute: MD Anderson Cancer Center

data_processing:
Illumina RTA v2.4.11 software was used for basecalling and bcl2fastq v2.17.14 was used for generating fastq files.
Sequenced reads were trimmed for adaptor sequence and low quality, then mapped to mm10 whole genome using STAR v2.6.1a with parameters with default settings.
Reads mapped to annotated genes were counted and aggregated using HTSeq_count v0.6.1.
Differential gene expression was analyzed using R package DESeq2.
Assembly: mm10
Supplementary files format and content: annotated gene expression count
Supplementary files format and content: Raw_ Counts

organism: Mus musculus

source_name: Pancreas

extract_protocol:
Pancreas were removed, RNA was harvested using Trizol reagent with pooled three mice pancreas, and  100 ng of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: Pancreas

tag: age

value: 2 month

tag: genotype

value: HPK1-M46 transgenic


title: G2 control rep1 [HWr_HPK1_13868]

value: B control


title: inf_BMDMs 1 scPAIR-seq


title: G3 HPK1-M46 rep2 [HWr_HPK1_13013]


title: G3 HPK1-M46 rep1 [HWr_HPK1_13458]


title: inf_BMDMs 4 scPAIR-seq


title: total RNAPII ChIP hyl1-2

city: Poznan

first: Jakub

middle: Jacek

last: Dolata

address: Uniwersytetu Poznanskiego 6

department: Department of Gene Expression

country: Poland

institute: Adam Mickiewicz University

library_source: genomic

data_processing:
Reads were trimmed to 50bp and aligned to the Arabidopsis genome (TAIR10) using Bowtie. Duplicate reads were removed by SAMtools. The remaining sequences were extended to 200bp according to ChIP fragment length. Plots of RNA Pol II distribution were made using ngs.plot software. Bed files were generated (Bedtools) to count reads for particular genomic regions.
Assembly: tair10

molecule: genomic DNA

organism: Arabidopsis thaliana

source_name: leaves

growth_protocol: temp 22C, long day, plants were grown on soil

extract_protocol:
Chromatin immunoprecipitation was performed using nuclei isolated from crosslinked (1% formaldehyde) 21-day old leaves, as described in (Bowler, Benvenuto et al. 2004) with minor modifications. Sonic and IP buffer were prepared as described in (Kaufmann, Muino et al. 2010). Chromatin was sonicated at 4°C with a Diagenode Bioruptor Plus at high intensity for 30 min (30 s on/30 s off) to obtain 200-300 bp DNA fragment size. Antibodies against total RNA Pol II (Abcam ab817) were used with Dynabeads Protein G (Thermo Scientific). Protein– DNA complexes were eluted from the beads as described in (Rowley, Bohmdorfer et al. 2013) and DNA was purified using a column-based method.
MicroPlex Library Preparation Kit v2 (Diagenode),

value: leaves

tag: chip antibody

value: anti-RNA Pol II (Abcam ab817)


title: G2 control rep2 [HWr_HPK1_13900]


title: G2 control rep3 [HWr_HPK1_13065]


title: total RNAPII ChIP WT Col-0

value: WT


title: total RNAPII ChIP WT Col-0 (input sample)

value: none (input)


title: PESA Study participant mRNA  SUAZ_511_v1

city: Madrid

first: Juan

middle: Carlos

last: Silla-Castro

address: Melchor Fernandez Almagro 3

department: Bioinformatics

country: Spain

institute: CNIC

data_processing:
Sequenced reads trimming for adaptors with cutadapt v.1.1.1
Quantification from identified transcripts using RSEM v1.2.20, which internally made alignment of reads against human genome reference GRCh38.82 using bowtie2 (30) v2.2.5.
Expected counts from each sequencing run were then joined into a table with 43,186 genes. Genes with expression value in CPM>1 for almost 80% of participants were selected and 12,062 genes were left for normalization with the TMM method implemented in EdgeR
Assembly: GRCh38.82
Supplementary files format and content: Tab delimited format with ensemble gene ID, gene name and participant’s RSEM expected counts.
Supplementary files format and content: Tab delimited format with ensemble gene ID, gene name and participant’s TMM normalized expression value for genes with CPM>1 for almost 80% of participants.

organism: Homo sapiens

source_name: WBS

extract_protocol:
Peripheral blood samples were collected from 391 PESA participants, transferred to PAXgene Blood RNA tubes (Preanalytix) to allow instant preservation of RNA, and stored at -80ºC until use. Total cellular RNA, including miRNA, was automatically extracted from PAXgene tubes using a QIAsymphony SP liquid handling robot (Qiagen) and the QIAsymphony PAXgene blood RNA kit (Qiagen), following the manufacturer´s instructions. RNA quantity was measured by absorbance using a NanoQuant Plate (Tecan). The RNA integrity number (RIN) was calculated by automated electrophoresis in a 2100 Bioanalyzer (RNA6000 Nano LabChip, Agilent).
Barcoded RNA-seq libraries were generated using 200 ng of total RNA with RIN>6 and the NEBNext Ultra RNA Library preparation kit (New England Biolabs). Briefly, polyA+ RNA was purified using polyT oligo-attached magnetic beads followed by fragmentation, and subsequent first and second cDNA strand synthesis. cDNA 3’ ends were adenylated, the adapters were ligated, and the library was amplified by PCR. The size and concentration of the libraries were checked using the Agilent 2100 Bioanalyzer DNA 1000 chip. Pools were prepared containing on average 11 libraries with a different barcode and equimolar concentrations of each library. Each pool concentration was determined using the Qubit® fluorometer (Life Technologies). Library pools were sequenced on a HiSeq2500 (Illumina) to generate 60 bases single reads. Fastq files for each sample were obtained using CASAVA v1.8 software (Illumina).

value: WBS

tag: gender

value: Male

tag: pesa score

value: Intermediate


title: PESA Study participant mRNA  SUAZ_508_v1

value: No


title: PESA Study participant mRNA  SUAZ_505_v1

value: Generalized


title: primary human epididymis epithelial cells, donor 1, scRNAseq

city: Cleveland

first: Ann

last: Harris

state: OH

address: 2109 Adelbert Road

department: Genetics

institute: Case Western Reserve University

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Demultiplexing, barcoded processing, gene counting, and aggregation were made using Cell Ranger v3.1.0
Assembly: hg19
Supplementary files format and content: Tab-separated values files and matrix files

molecule: polyA RNA

growth_protocol: Normal (i.e. no tumor invasion) epididymis tissue was collected within ~ 2 hr of orchiectomy surgery from patients with a clinical diagnosis of testicular cancer.  Caput tissue was cut into 1-2 mm pieces and digested with collagenase as detailed below. Human epididymis epithelial cells were grown on Primaria plastic (Corning) in CMRL-1066 media (containing 15% fetal calf serum, 2 mmol/L L-glutamine, 1 μg/mL hydrocortisone, 0.2 U/mL insulin, and 10−10 mol/L cholera toxin) as described in DOI: 10.1016/j.fertnstert.2014.11.022

extract_protocol:
Normal (i.e. no tumor invasion) epididymis tissue was collected within ~ 2 hr of orchiectomy surgery from patients with a clinical diagnosis of testicular cancer.  Efferent duct and caput tissue was cut into 1-2 mm pieces and digested with collagenase (2 mg/mL collagenase + 40 U/mL DNAse I, Worthington Biochemical Corp.) at 37°C for 2 hr with constant shaking. Digesting tissue was then agitated by pipetting, allowed to settle for 4 min and the supernatant collected and stored on ice as cell suspension 1. Fresh collagenase solution was then added to the tissue, which was digested for a further 2 hr with shaking at 37°C, with subsequent agitation and settling as before. The supernatant (cell suspension 2) was then pooled with cell suspension 1 and tissue debris removed by passage through a 100-µm cell strainer (Pluristrainer). Following centrifugation of the cells (300×g at room temperature) they were washed once in PBS and then digested with Accutase (STEMCELL Technologies) for 20-30 min at 37°C to obtain single cell suspension. After centrifugation, the cell pellet was lysed with ammonium chloride solution (310 mM NH4Cl, 23.8 mM NaHCO3 and 0.2 mM EDTA) for 3 min followed by adding 2.5× volume of PBS + 2% fetal bovine serum (FBS) and centrifuged at 300×g for 5 min (at room temperature) to remove red blood cells. Epithelial cells collected were washed twice in Hank's Balanced Salt Solution (HBSS) + 2% FBS with centrifugation at (200×g at 4°C), then resuspended in HBSS and passed through Flowmi cell strainers (40 µm, Bel-Art) to prepare single cells. 3000 cells were used for scRNA-seq using the 10x Genomics Chromium system.
Libraries generated from single cells according to the manufacturer's protocol for 10X Genomics Chromium Single Cell 3' Reagent Kits v2 (for AXH005 and AXH007) or 10X Genomics Chromium Single Cell 3' Reagent Kits v3 (for AXH019 and AXH020)

tag: cell type

tag: Sex

value: male

tag: disease state

value: normal


title: PESA Study participant mRNA  SUAZ_510_v1


title: PESA Study participant mRNA  SUAZ_506_v1


title: PESA Study participant mRNA  SUAZ_512_v1


title: PESA Study participant mRNA  SUAZ_500_v1


title: PESA Study participant mRNA  SUAZ_509_v1


title: PESA Study participant mRNA  SUAZ_515_v1


title: PESA Study participant mRNA  SUAZ_516_v1


title: PESA Study participant mRNA  SUAZ_502_v1


title: PESA Study participant mRNA  SUAZ_499_v1


title: PESA Study participant mRNA  SUAZ_504_v1


title: PESA Study participant mRNA  SUAZ_497_v1


title: PESA Study participant mRNA  SUAZ_514_v1


title: PESA Study participant mRNA  SUAZ_503_v1

value: Focal


title: PESA Study participant mRNA  SUAZ_507_v1


title: PESA Study participant mRNA  SUAZ_492_v1


title: PESA Study participant mRNA  SUAZ_491_v1


title: PESA Study participant mRNA  SUAZ_490_v1


title: total RNAPII ChIP hyl1-2 (input sample)


title: PESA Study participant mRNA  SUAZ_483_v1


title: PESA Study participant mRNA  SUAZ_489_v1


title: PESA Study participant mRNA  SUAZ_480_v1


title: PESA Study participant mRNA  SUAZ_479_v1


title: PESA Study participant mRNA  SUAZ_481_v1


title: PESA Study participant mRNA  SUAZ_478_v1


title: PESA Study participant mRNA  SUAZ_498_v1


title: PESA Study participant mRNA  SUAZ_473_v1


title: PESA Study participant mRNA  SUAZ_476_v1


title: PESA Study participant mRNA  SUAZ_477_v1


title: PESA Study participant mRNA  SUAZ_472_v1


title: PESA Study participant mRNA  SUAZ_482_v1


title: PESA Study participant mRNA  SUAZ_475_v1


title: PESA Study participant mRNA  SUAZ_464_v1


title: PESA Study participant mRNA  SUAZ_455_v1


title: PESA Study participant mRNA  SUAZ_460_v1


title: PESA Study participant mRNA  SUAZ_468_v1


title: PESA Study participant mRNA  SUAZ_459_v1


title: PESA Study participant mRNA  SUAZ_453_v1


title: PESA Study participant mRNA  SUAZ_465_v1


title: PESA Study participant mRNA  SUAZ_448_v1


title: PESA Study participant mRNA  SUAZ_449_v1


title: PESA Study participant mRNA  SUAZ_462_v1


title: PESA Study participant mRNA  SUAZ_446_v1


title: PESA Study participant mRNA  SUAZ_447_v1


title: PESA Study participant mRNA  SUAZ_444_v1


title: PESA Study participant mRNA  SUAZ_456_v1


title: PESA Study participant mRNA  SUAZ_470_v1


title: PESA Study participant mRNA  SUAZ_452_v1


title: PESA Study participant mRNA  SUAZ_445_v1


title: PESA Study participant mRNA  SUAZ_463_v1


title: PESA Study participant mRNA  SUAZ_457_v1


title: PESA Study participant mRNA  SUAZ_450_v1


title: PESA Study participant mRNA  SUAZ_471_v1


title: PESA Study participant mRNA  SUAZ_443_v1


title: PESA Study participant mRNA  SUAZ_454_v1


title: PESA Study participant mRNA  SUAZ_451_v1


title: PESA Study participant mRNA  SUAZ_441_v1


title: PESA Study participant mRNA  SUAZ_440_v1


title: PESA Study participant mRNA  SUAZ_439_v1


title: PESA Study participant mRNA  SUAZ_437_v1


title: PESA Study participant mRNA  SUAZ_438_v1


title: PESA Study participant mRNA  SUAZ_436_v1


title: PESA Study participant mRNA  SUAZ_429_v1


title: PESA Study participant mRNA  SUAZ_430_v1


title: PESA Study participant mRNA  SUAZ_431_v1


title: PESA Study participant mRNA  SUAZ_418_v1


title: PESA Study participant mRNA  SUAZ_425_v1


title: PESA Study participant mRNA  SUAZ_423_v1


title: PESA Study participant mRNA  SUAZ_420_v1


title: PESA Study participant mRNA  SUAZ_417_v1


title: PESA Study participant mRNA  SUAZ_424_v1


title: PESA Study participant mRNA  SUAZ_421_v1


title: PESA Study participant mRNA  SUAZ_419_v1


title: PESA Study participant mRNA  SUAZ_422_v1


title: PESA Study participant mRNA  SUAZ_413_v1


title: PESA Study participant mRNA  SUAZ_414_v1


title: PESA Study participant mRNA  SUAZ_432_v1


title: PESA Study participant mRNA  SUAZ_428_v1


title: PESA Study participant mRNA  SUAZ_415_v1


title: PESA Study participant mRNA  SUAZ_416_v1


title: PESA Study participant mRNA  SUAZ_426_v1


title: PESA Study participant mRNA  SUAZ_412_v1


title: PESA Study participant mRNA  SUAZ_411_v1


title: PESA Study participant mRNA  SUAZ_410_v1


title: PESA Study participant mRNA  SUAZ_406_v1


title: PESA Study participant mRNA  SUAZ_427_v1


title: PESA Study participant mRNA  SUAZ_405_v1


title: PESA Study participant mRNA  SUAZ_404_v1


title: PESA Study participant mRNA  SUAZ_433_v1


title: PESA Study participant mRNA  SUAZ_403_v1

value: Female


title: PESA Study participant mRNA  SUAZ_402_v1


title: PESA Study participant mRNA  SUAZ_399_v1


title: PESA Study participant mRNA  SUAZ_396_v1


title: PESA Study participant mRNA  SUAZ_393_v1


title: PESA Study participant mRNA  SUAZ_409_v1


title: PESA Study participant mRNA  SUAZ_400_v1


title: PESA Study participant mRNA  SUAZ_391_v1


title: PESA Study participant mRNA  SUAZ_394_v1


title: PESA Study participant mRNA  SUAZ_382_v1


title: PESA Study participant mRNA  SUAZ_398_v1


title: PESA Study participant mRNA  SUAZ_383_v1


title: PESA Study participant mRNA  SUAZ_380_v1


title: PESA Study participant mRNA  SUAZ_387_v1


title: PESA Study participant mRNA  SUAZ_384_v1


title: PESA Study participant mRNA  SUAZ_407_v1


title: PESA Study participant mRNA  SUAZ_390_v1


title: PESA Study participant mRNA  SUAZ_385_v1


title: PESA Study participant mRNA  SUAZ_376_v1


title: PESA Study participant mRNA  SUAZ_377_v1


title: PESA Study participant mRNA  SUAZ_375_v1


title: PESA Study participant mRNA  SUAZ_401_v1


title: PESA Study participant mRNA  SUAZ_372_v1


title: PESA Study participant mRNA  SUAZ_388_v1


title: PESA Study participant mRNA  SUAZ_392_v1


title: PESA Study participant mRNA  SUAZ_395_v1


title: PESA Study participant mRNA  SUAZ_371_v1


title: PESA Study participant mRNA  SUAZ_370_v1


title: PESA Study participant mRNA  SUAZ_369_v1


title: PESA Study participant mRNA  SUAZ_379_v1


title: PESA Study participant mRNA  SUAZ_367_v1


title: PESA Study participant mRNA  SUAZ_365_v1


title: PESA Study participant mRNA  SUAZ_368_v1


title: PESA Study participant mRNA  SUAZ_366_v1


title: PESA Study participant mRNA  SUAZ_364_v1


title: PESA Study participant mRNA  SUAZ_362_v1


title: PESA Study participant mRNA  SUAZ_360_v1


title: PESA Study participant mRNA  SUAZ_361_v1


title: PESA Study participant mRNA  SUAZ_357_v1


title: PESA Study participant mRNA  SUAZ_356_v1


title: PESA Study participant mRNA  SUAZ_358_v1


title: PESA Study participant mRNA  SUAZ_363_v1


title: PESA Study participant mRNA  SUAZ_355_v1


title: PESA Study participant mRNA  SUAZ_354_v1


title: PESA Study participant mRNA  SUAZ_352_v1


title: PESA Study participant mRNA  SUAZ_353_v1


title: PESA Study participant mRNA  SUAZ_351_v1


title: PESA Study participant mRNA  SUAZ_347_v1


title: PESA Study participant mRNA  SUAZ_349_v1


title: PESA Study participant mRNA  SUAZ_346_v1


title: PESA Study participant mRNA  SUAZ_345_v1


title: PESA Study participant mRNA  SUAZ_344_v1


title: PESA Study participant mRNA  SUAZ_342_v1


title: PESA Study participant mRNA  SUAZ_343_v1


title: PESA Study participant mRNA  SUAZ_339_v1


title: PESA Study participant mRNA  SUAZ_359_v1


title: PESA Study participant mRNA  SUAZ_338_v1


title: PESA Study participant mRNA  SUAZ_442_v1


title: PESA Study participant mRNA  SUAZ_335_v1


title: PESA Study participant mRNA  SUAZ_333_v1


title: PESA Study participant mRNA  SUAZ_337_v1


title: PESA Study participant mRNA  SUAZ_332_v1


title: PESA Study participant mRNA  SUAZ_331_v1


title: PESA Study participant mRNA  SUAZ_328_v1


title: PESA Study participant mRNA  SUAZ_329_v1


title: PESA Study participant mRNA  SUAZ_326_v1


title: PESA Study participant mRNA  SUAZ_324_v1


title: PESA Study participant mRNA  SUAZ_327_v1


title: PESA Study participant mRNA  SUAZ_325_v1


title: PESA Study participant mRNA  SUAZ_320_v1


title: PESA Study participant mRNA  SUAZ_322_v1


title: PESA Study participant mRNA  SUAZ_317_v1


title: PESA Study participant mRNA  SUAZ_316_v1


title: PESA Study participant mRNA  SUAZ_323_v1


title: PESA Study participant mRNA  SUAZ_315_v1


title: PESA Study participant mRNA  SUAZ_313_v1


title: PESA Study participant mRNA  SUAZ_340_v1


title: PESA Study participant mRNA  SUAZ_314_v1


title: PESA Study participant mRNA  SUAZ_312_v1


title: PESA Study participant mRNA  SUAZ_318_v1


title: PESA Study participant mRNA  SUAZ_310_v1


title: PESA Study participant mRNA  SUAZ_330_v1


title: PESA Study participant mRNA  SUAZ_319_v1


title: PESA Study participant mRNA  SUAZ_301_v1


title: PESA Study participant mRNA  SUAZ_309_v1


title: PESA Study participant mRNA  SUAZ_304_v1


title: PESA Study participant mRNA  SUAZ_307_v1


title: PESA Study participant mRNA  SUAZ_305_v1


title: PESA Study participant mRNA  SUAZ_303_v1


title: PESA Study participant mRNA  SUAZ_308_v1


title: PESA Study participant mRNA  SUAZ_306_v1


title: PESA Study participant mRNA  SUAZ_295_v1


title: PESA Study participant mRNA  SUAZ_299_v1


title: PESA Study participant mRNA  SUAZ_293_v1


title: PESA Study participant mRNA  SUAZ_296_v1


title: PESA Study participant mRNA  SUAZ_298_v1


title: PESA Study participant mRNA  SUAZ_292_v1


title: PESA Study participant mRNA  SUAZ_291_v1


title: PESA Study participant mRNA  SUAZ_290_v1


title: PESA Study participant mRNA  SUAZ_289_v1


title: PESA Study participant mRNA  SUAZ_288_v1


title: PESA Study participant mRNA  SUAZ_311_v1


title: PESA Study participant mRNA  SUAZ_286_v1


title: PESA Study participant mRNA  SUAZ_287_v1


title: PESA Study participant mRNA  SUAZ_283_v1


title: PESA Study participant mRNA  SUAZ_284_v1


title: PESA Study participant mRNA  SUAZ_282_v1


title: PESA Study participant mRNA  SUAZ_281_v1


title: PESA Study participant mRNA  SUAZ_302_v1


title: PESA Study participant mRNA  SUAZ_300_v1


title: PESA Study participant mRNA  SUAZ_279_v1


title: PESA Study participant mRNA  SUAZ_275_v1


title: PESA Study participant mRNA  SUAZ_277_v1


title: PESA Study participant mRNA  SUAZ_271_v1


title: PESA Study participant mRNA  SUAZ_274_v1


title: PESA Study participant mRNA  SUAZ_278_v1


title: PESA Study participant mRNA  SUAZ_272_v1


title: PESA Study participant mRNA  SUAZ_273_v1


title: PESA Study participant mRNA  SUAZ_276_v1


title: PESA Study participant mRNA  SUAZ_268_v1


title: PESA Study participant mRNA  SUAZ_270_v1


title: PESA Study participant mRNA  SUAZ_262_v1


title: PESA Study participant mRNA  SUAZ_269_v1


title: PESA Study participant mRNA  SUAZ_257_v1


title: PESA Study participant mRNA  SUAZ_267_v1


title: PESA Study participant mRNA  SUAZ_263_v1


title: PESA Study participant mRNA  SUAZ_285_v1


title: PESA Study participant mRNA  SUAZ_259_v1


title: PESA Study participant mRNA  SUAZ_255_v1


title: PESA Study participant mRNA  SUAZ_254_v1


title: PESA Study participant mRNA  SUAZ_253_v1


title: PESA Study participant mRNA  SUAZ_252_v1


title: PESA Study participant mRNA  SUAZ_251_v1


title: PESA Study participant mRNA  SUAZ_250_v1


title: PESA Study participant mRNA  SUAZ_249_v1


title: PESA Study participant mRNA  SUAZ_247_v1


title: PESA Study participant mRNA  SUAZ_248_v1


title: PESA Study participant mRNA  SUAZ_246_v1


title: PESA Study participant mRNA  SUAZ_245_v1


title: PESA Study participant mRNA  SUAZ_266_v1


title: PESA Study participant mRNA  SUAZ_243_v1


title: PESA Study participant mRNA  SUAZ_241_v1


title: PESA Study participant mRNA  SUAZ_238_v1


title: PESA Study participant mRNA  SUAZ_236_v1


title: PESA Study participant mRNA  SUAZ_240_v1


title: PESA Study participant mRNA  SUAZ_230_v1


title: PESA Study participant mRNA  SUAZ_237_v1


title: PESA Study participant mRNA  SUAZ_239_v1


title: PESA Study participant mRNA  SUAZ_229_v1


title: PESA Study participant mRNA  SUAZ_228_v1


title: PESA Study participant mRNA  SUAZ_223_v1


title: PESA Study participant mRNA  SUAZ_222_v1


title: PESA Study participant mRNA  SUAZ_227_v1


title: PESA Study participant mRNA  SUAZ_215_v1


title: PESA Study participant mRNA  SUAZ_221_v1


title: PESA Study participant mRNA  SUAZ_217_v1


title: PESA Study participant mRNA  SUAZ_226_v1


title: PESA Study participant mRNA  SUAZ_218_v1


title: PESA Study participant mRNA  SUAZ_216_v1


title: PESA Study participant mRNA  SUAZ_212_v1


title: PESA Study participant mRNA  SUAZ_208_v1


title: PESA Study participant mRNA  SUAZ_205_v1


title: PESA Study participant mRNA  SUAZ_213_v1


title: PESA Study participant mRNA  SUAZ_203_v1


title: PESA Study participant mRNA  SUAZ_233_v1


title: PESA Study participant mRNA  SUAZ_234_v1


title: PESA Study participant mRNA  SUAZ_204_v1


title: PESA Study participant mRNA  SUAZ_209_v1


title: PESA Study participant mRNA  SUAZ_202_v1


title: PESA Study participant mRNA  SUAZ_200_v1


title: PESA Study participant mRNA  SUAZ_192_v1


title: PESA Study participant mRNA  SUAZ_195_v1


title: PESA Study participant mRNA  SUAZ_196_v1


title: PESA Study participant mRNA  SUAZ_189_v1


title: PESA Study participant mRNA  SUAZ_191_v1


title: PESA Study participant mRNA  SUAZ_190_v1


title: PESA Study participant mRNA  SUAZ_188_v1


title: PESA Study participant mRNA  SUAZ_197_v1


title: PESA Study participant mRNA  SUAZ_185_v1


title: PESA Study participant mRNA  SUAZ_186_v1


title: PESA Study participant mRNA  SUAZ_184_v1


title: PESA Study participant mRNA  SUAZ_211_v1


title: PESA Study participant mRNA  SUAZ_187_v1


title: PESA Study participant mRNA  SUAZ_179_v1


title: PESA Study participant mRNA  SUAZ_181_v1


title: PESA Study participant mRNA  SUAZ_183_v1


title: PESA Study participant mRNA  SUAZ_177_v1


title: PESA Study participant mRNA  SUAZ_176_v1


title: PESA Study participant mRNA  SUAZ_175_v1


title: PESA Study participant mRNA  SUAZ_199_v1


title: PESA Study participant mRNA  SUAZ_170_v1


title: PESA Study participant mRNA  SUAZ_172_v1


title: PESA Study participant mRNA  SUAZ_171_v1


title: PESA Study participant mRNA  SUAZ_169_v1


title: PESA Study participant mRNA  SUAZ_167_v1


title: PESA Study participant mRNA  SUAZ_163_v1


title: PESA Study participant mRNA  SUAZ_161_v1


title: PESA Study participant mRNA  SUAZ_160_v1


title: PESA Study participant mRNA  SUAZ_157_v1


title: PESA Study participant mRNA  SUAZ_155_v1


title: PESA Study participant mRNA  SUAZ_180_v1


title: PESA Study participant mRNA  SUAZ_159_v1


title: PESA Study participant mRNA  SUAZ_374_v1


title: PESA Study participant mRNA  SUAZ_154_v1


title: PESA Study participant mRNA  SUAZ_152_v1


title: PESA Study participant mRNA  SUAZ_151_v1


title: PESA Study participant mRNA  SUAZ_148_v1


title: PESA Study participant mRNA  SUAZ_149_v1


title: PESA Study participant mRNA  SUAZ_147_v1


title: PESA Study participant mRNA  SUAZ_145_v1


title: PESA Study participant mRNA  SUAZ_150_v1


title: PESA Study participant mRNA  SUAZ_173_v1


title: PESA Study participant mRNA  SUAZ_142_v1


title: PESA Study participant mRNA  SUAZ_143_v1


title: PESA Study participant mRNA  SUAZ_144_v1


title: PESA Study participant mRNA  SUAZ_164_v1


title: PESA Study participant mRNA  SUAZ_140_v1


title: PESA Study participant mRNA  SUAZ_141_v1


title: PESA Study participant mRNA  SUAZ_139_v1


title: PESA Study participant mRNA  SUAZ_138_v1


title: PESA Study participant mRNA  SUAZ_135_v1


title: PESA Study participant mRNA  SUAZ_130_v1


title: PESA Study participant mRNA  SUAZ_156_v1


title: PESA Study participant mRNA  SUAZ_153_v1


title: PESA Study participant mRNA  SUAZ_127_v1


title: PESA Study participant mRNA  SUAZ_129_v1


title: PESA Study participant mRNA  SUAZ_126_v1


title: PESA Study participant mRNA  SUAZ_116_v1


title: PESA Study participant mRNA  SUAZ_146_v1


title: PESA Study participant mRNA  SUAZ_123_v1


title: PESA Study participant mRNA  SUAZ_112_v1


title: PESA Study participant mRNA  SUAZ_111_v1


title: PESA Study participant mRNA  SUAZ_122_v1


title: PESA Study participant mRNA  SUAZ_115_v1


title: PESA Study participant mRNA  SUAZ_110_v1


title: PESA Study participant mRNA  SUAZ_104_v1


title: PESA Study participant mRNA  SUAZ_106_v1


title: PESA Study participant mRNA  SUAZ_108_v1


title: PESA Study participant mRNA  SUAZ_098_v1


title: PESA Study participant mRNA  SUAZ_103_v1


title: PESA Study participant mRNA  SUAZ_097_v1


title: PESA Study participant mRNA  SUAZ_096_v1


title: PESA Study participant mRNA  SUAZ_095_v1


title: PESA Study participant mRNA  SUAZ_093_v1


title: PESA Study participant mRNA  SUAZ_092_v1


title: PESA Study participant mRNA  SUAZ_090_v1


title: PESA Study participant mRNA  SUAZ_089_v1


title: PESA Study participant mRNA  SUAZ_088_v1


title: PESA Study participant mRNA  SUAZ_094_v1


title: PESA Study participant mRNA  SUAZ_124_v1


title: PESA Study participant mRNA  SUAZ_085_v1


title: PESA Study participant mRNA  SUAZ_114_v1


title: PESA Study participant mRNA  SUAZ_084_v1


title: PESA Study participant mRNA  SUAZ_077_v1


title: PESA Study participant mRNA  SUAZ_075_v1


title: PESA Study participant mRNA  SUAZ_078_v1


title: PESA Study participant mRNA  SUAZ_072_v1


title: PESA Study participant mRNA  SUAZ_071_v1


title: PESA Study participant mRNA  SUAZ_067_v1


title: PESA Study participant mRNA  SUAZ_062_v1


title: PESA Study participant mRNA  SUAZ_063_v1


title: PESA Study participant mRNA  SUAZ_058_v1


title: PESA Study participant mRNA  SUAZ_086_v1


title: PESA Study participant mRNA  SUAZ_057_v1


title: PESA Study participant mRNA  SUAZ_056_v1


title: PESA Study participant mRNA  SUAZ_059_v1


title: PESA Study participant mRNA  SUAZ_053_v1


title: PESA Study participant mRNA  SUAZ_054_v1


title: PESA Study participant mRNA  SUAZ_081_v1


title: PESA Study participant mRNA  SUAZ_047_v1


title: PESA Study participant mRNA  SUAZ_049_v1


title: PESA Study participant mRNA  SUAZ_046_v1


title: PESA Study participant mRNA  SUAZ_048_v1


title: PESA Study participant mRNA  SUAZ_044_v1


title: PESA Study participant mRNA  SUAZ_038_v1


title: PESA Study participant mRNA  SUAZ_079_v1


title: PESA Study participant mRNA  SUAZ_039_v1


title: PESA Study participant mRNA  SUAZ_034_v1


title: PESA Study participant mRNA  SUAZ_033_v1


title: PESA Study participant mRNA  SUAZ_027_v1


title: PESA Study participant mRNA  SUAZ_031_v1


title: PESA Study participant mRNA  SUAZ_029_v1


title: PESA Study participant mRNA  SUAZ_025_v1


title: PESA Study participant mRNA  SUAZ_024_v1


title: PESA Study participant mRNA  SUAZ_022_v1


title: PESA Study participant mRNA  SUAZ_023_v1


title: PESA Study participant mRNA  SUAZ_020_v1


title: PESA Study participant mRNA  SUAZ_014_v1


title: PESA Study participant mRNA  SUAZ_009_v1


title: PESA Study participant mRNA  SUAZ_021_v1


title: PESA Study participant mRNA  SUAZ_007_v1


title: PESA Study participant mRNA  SUAZ_008_v1


title: PESA Study participant mRNA  SUAZ_016_v1


title: PESA Study participant mRNA  SUAZ_017_v1


title: PESA Study participant mRNA  SUAZ_005_v1


title: 23777_MLH1-TEP-D30-2_S36

city: Duarte

first: wei

last: chen

address: 1500 E Duarte Rd

department: Cancer Biology-BRI

institute: City of Hope National Medical Center

data_processing:
Base calling was done using Illumina RTA and bcl2fastq v2.18.0.12
Sequences were aligned to hg19 using Tophat2 with default settings
Gene expression level of Ensembl GRCh37 coding genes were quantified using HTSeq v0.6.1p1
Assembly: hg19
Supplementary files format and content: tab delimited text file gene expression counts

source_name: H1 PM-MLH1 hESCs-D30 TECs

extract_protocol:
Qiagen blood and tissue kit
KAPA mRNA HyperPrep Kits
Enrich mRNA in samples and provide a focused view of the protein-coding regions in the transcriptome.

treatment_protocol: (+/-) MLH1 DNA methylation and differentiation into cardiomyocyte or thymic epthelial cell lineage

tag: cell line

value: H1 PM-MLH1 hESCs-D30 TECs

value: thymic epithelial cell

tag: treatment

value: TEC Differentiation- HOXA3/FOXN1


title: 23773_MLH1-TEP-D10-2_S32

source_name: H1 PM-MLH1 hESCs-D10 differentiation to TECs

value: differentiating cell


title: 23775_MLH1-TEP-D20-2_S34

source_name: H1 PM-MLH1 hESCs-D20 differentiation to TECs


title: 23776_MLH1-TEP-D30-1_S35


title: 23774_MLH1-TEP-D20-1_S33

value: H1 PM-MLH1 hESCs-D20 differentiation to TECs


title: 23771_MLH1-TEP-D4-2_S30

source_name: H1 PM-MLH1 hESCs-D4 differentiation to TECs


title: 23772_MLH1-TEP-D10-1_S31

value: H1 PM-MLH1 hESCs-D10 differentiation to TECs


title: PESA Study participant mRNA  SUAZ_001_v1


title: 23770_MLH1-TEP-D4-1_S29

value: H1 PM-MLH1 hESCs-D4 differentiation to TECs


title: 23768_TEP-D30-1_S27

source_name: H1 hESCs-D30 differentiation to TECs

value: H1 hESCs-D30 differentiation to TECs

value: WT


title: 23767_TEP-D20-2_S26

source_name: H1 hESCs-D20 differentiation to TECs

value: H1 hESCs-D20 differentiation to TECs


title: 23764_TEP-D10-1_S23

source_name: H1 hESCs-D10 differentiation to TECs

value: H1 hESCs-D10 differentiation to TECs


title: 23763_TEP-D4-2_S22

source_name: H1 hESCs-D4 differentiation to TECs

value: H1 hESCs-D4 differentiation to TECs


title: PESA Study participant mRNA  SUAZ_013_v1

value: WBS

value: Male

value: No


title: 23762_TEP-D4-1_S21


title: 23760_H1M6-CM-D21-1_S19

source_name: H1 PM-MLH1  derived cardiomyocytes (D21)

value: H1 PM-MLH1  derived cardiomyocytes (D21)

value: cardiomyocyte

value: CM Differentiation- IWP4/CHIR99025


title: 23761_H1M6-CM-D21-2_S20

source_name: H1 PM-MLH1 derived cardiomyocytes (D21)

value: H1 PM-MLH1 derived cardiomyocytes (D21)

value: CM Differentiation- IWP4/CHIR99026


title: PESA Study participant mRNA  SUAZ_006_v1


title: 23759_H1M6-CM-D10-2_S18

source_name: H1 PM-MLH1 hESCs-D10 differentiation to CMs

value: differentiating cell


title: 23758_H1M6-CM-D10-1_S17

value: H1 PM-MLH1 hESCs-D10 differentiation to CMs


title: 23757_H1M6-CM-D4-2_S16

source_name: H1 PM-MLH1 hESCs-D4 differentiation to CMs

value: CM Differentiation- IWP4/CHIR99024


title: 23756_H1M6-CM-D4-1_S15

value: H1 PM-MLH1 hESCs-D4 differentiation to CMs

value: CM Differentiation- IWP4/CHIR99023


title: 23755_H1M6-CM-D2-2_S14

source_name: H1 PM-MLH1 hESCs-D2 differentiation to CMs

value: CM Differentiation- IWP4/CHIR99022


title: 23754_H1M6-CM-D2-1_S13

value: H1 PM-MLH1 hESCs-D2 differentiation to CMs

value: CM Differentiation- IWP4/CHIR99021


title: 23753_H1M6-CM-D0-2_S12

source_name: H1-PM-MLH1 hESCs

value: H1-PM-MLH1 hESCs

value: pluripotent stem cell


title: 23751_H1-CM-D21-2_S10

source_name: H1 derived cardiomyocytes (D21)

value: H1 derived cardiomyocytes (D21)

value: WT


title: 23746_H1-CM-D4-1_S5

source_name: H1 hESCs-D4 differentiation to CMs

value: H1 hESCs-D4 differentiation to CMs


title: 23748_H1-CM-D10-1_S7

source_name: H1 hESCs-D10 differentiation to CMs

value: H1 hESCs-D10 differentiation to CMs


title: 23745_H1-CM-D2-2_S4

source_name: H1 hESCs-D2 differentiation to CMs

value: H1 hESCs-D2 differentiation to CMs


title: 23766_TEP-D20-1_S25

value: H1 hESCs-D20 differentiation to TECs

value: TEC Differentiation- HOXA3/FOXN1


title: 23752_H1M6-CM-D0-1_S11


title: 23769_TEP-D30-2_S28

value: H1 hESCs-D30 differentiation to TECs

value: thymic epithelial cell


title: 23749_H1-CM-D10-2_S8

value: differentiating cell

value: CM Differentiation- IWP4/CHIR99026


title: 23747_H1-CM-D4-2_S6

value: CM Differentiation- IWP4/CHIR99024


title: 23765_TEP-D10-2_S24

value: H1 hESCs-D10 differentiation to TECs


title: 23744_H1-CM-D2-1_S3


title: 23743_H1-CM-D0-2_S2

source_name: H1 hESCs

value: H1 hESCs


title: Day 10 - Diff. medium rep2 pat. 2

city: Nantes

first: Benjamin

last: Ory

address: 1 rue Gaston Veil

country: France

data_processing:
ChIP-Seq: reads were aligned to human genome (hg19) using bwa mem (v. 0.7.17). Low quality reads (-q 20), PCR duplicates and non-uniquely aligned reads using samtools (v. 1.9). Inorder to remove repliacte specific signals (alignement files of replicates from the same donor were merged into one alignement file using samtools merge. Peak calling was then performed on the merged alignement files using recommendations from github page of MACS2 (https://github.com/taoliu/MACS/wiki/Advanced\%3A-Call-peaks-using-MACS2-subcommands). Using the recommandations, ChIP and input tracks were built separately and then broad peak calling was perfomed using q-value threshold of 1e-5. ROSE2 was used to identify super-enhancers among peaks outside of promoters (TSS +/-2500 b.p.).
Assembly: hg19
Supplementary files format and content: comma-delimited text files per sample include all enhancers and super-enhancers identified, as output from ROSE2

source_name: PBMC

extract_protocol: ChIP-Seq: Cells were harvested, frozen and sent to Broad Institute’s Genomics Services platform for the ChIP data and to Active Motif Inc. platform for the input data. H3K27ac antibodies (Acetyl-Histone H3 K27 (D5E4) XR Rabbit mAB, Cell Signaling Technologies were used for precipitation. Library preparation and Sequencing for the ChIP-data were performed in a multiplexed system on a 96-well plate.

tag: subject status

value: healthy donor

value: PBMC

value: CD14+ pre-monocytes

value: Day 10 - Diff. medium


title: 23750_H1-CM-D21-1_S9

value: H1 derived cardiomyocytes (D21)

value: cardiomyocyte

value: WT

value: CM Differentiation- IWP4/CHIR99025


title: 23742_H1-CM-D0-1_S1

value: pluripotent stem cell


title: Day 10 - Diff. medium rep1 pat. 2

value: Acetyl-Histone H3 K27 (D5E4) XR Rabbit mAB, Cell Signaling Technologies


title: Day 10 - Diff. medium rep2 pat. 1


title: Day 10 - Diff. medium rep1 pat. 1


title: Day 7 - Diff. medium rep2 pat. 2

value: Day 7 - Diff. medium


title: Day 7 - Diff. medium rep2 pat. 1


title: Day 7 - Diff. medium rep1 pat. 2


title: Day 7 - Diff. medium rep1 pat. 1


title: Day 7 + Diff. medium rep2 pat. 2

description:
d7.+.diff_rep2_pat2
OC.J7.+.diff.rep2.pat.2_H3K27ac

data_processing:
RNA-Seq: FASTP was used for adpater trimming and to remove low-quality reads (Q>30). Reads were mapped to human genome (hg19) using HISAT2 (v. 2.1.0) and read quantification was performed using stringtie (v. 2.1.1).
ChIP-Seq: reads were aligned to human genome (hg19) using bwa mem (v. 0.7.17). Low quality reads (-q 20), PCR duplicates and non-uniquely aligned reads using samtools (v. 1.9). Inorder to remove repliacte specific signals (alignement files of replicates from the same donor were merged into one alignement file using samtools merge. Peak calling was then performed on the merged alignement files using recommendations from github page of MACS2 (https://github.com/taoliu/MACS/wiki/Advanced\%3A-Call-peaks-using-MACS2-subcommands). Using the recommandations, ChIP and input tracks were built separately and then broad peak calling was perfomed using q-value threshold of 1e-5. ROSE2 was used to identify super-enhancers among peaks outside of promoters (TSS +/-2500 b.p.).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files per sample include coverage, TPM, FPKM values for each gene.
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample.
Supplementary_files_format_and_content: comma-delimited text files per sample include all enhancers and super-enhancers identified, as output from ROSE2

source_name: Day 7 + Diff. medium_PBMC

extract_protocol:
RNA-Seq: Cells were harvested, frozen and sent to Active Motif Inc. platform for library preparation and sequencing. Illumina TruSeq Stranded mRNA Sample Preparation Kit was used for library preparation.
ChIP-Seq: Cells were harvested, frozen and sent to Broad Institute’s Genomics Services platform for the ChIP data and to Active Motif Inc. platform for the input data. H3K27ac antibodies (Acetyl-Histone H3 K27 (D5E4) XR Rabbit mAB, Cell Signaling Technologies were used for precipitation. Library preparation and Sequencing for the ChIP-data were performed in a multiplexed system on a 96-well plate.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Day 7 + Diff. medium


title: Day 7 + Diff. medium rep1 pat. 2

description:
d7.+.diff_rep1_pat2
OC.J7.+.diff.rep1.pat.2_H3K27ac


title: Day 7 + Diff. medium rep2 pat. 1

description:
d7.+.diff_rep2_pat1
OC.J7.+.diff.rep2.pat.1_H3K27ac


title: Day 7 + Diff. medium rep1 pat. 1

description:
D7.+.diff_rep1_pat1
OC.J7.+.diff.rep1.pat.1_H3K27ac


title: Day 3 - Diff. medium rep2 pat. 2

description:
D3.-.diff_rep2_pat2
OC.J3.rep2.pat.2_H3K27ac

source_name: Day 3 - Diff. medium_PBMC

value: Day 3 - Diff. medium


title: Day 3 - Diff. medium rep1 pat. 2

description:
d3.-.diff_rep1_pat2
OC.J3.rep1.pat.2_H3K27ac


title: Day 3 - Diff. medium rep2 pat. 1

description:
D3.-.diff_rep2_pat1
OC.J3.rep2.pat.1_H3K27ac


title: Day 10 + Diff. medium rep2 pat. 2

description:
d10.+.diff_rep2_pat2
OC.J10.+.rep2.pat.2_H3K27ac

source_name: Day 10 + Diff. medium_PBMC

value: Day 10 + Diff. medium


title: Day 10 + Diff. medium rep1 pat. 2

description:
d10.+.diff_rep1_pat2
OC.J10.+.rep1.pat.2_H3K27ac


title: Day 10 + Diff. medium rep1 pat. 1

description:
d10.+.diff_rep1_pat1
OC.J10.+.diff.rep1.pat.1_H3K27ac


title: Day 3 - Diff. medium rep1 pat. 1

description:
D3.-.diff_rep1_pat1
OC.J3.rep1.pat.1_H3K27ac


title: SPI2_mut_pool

data_processing:
Library strategy: PAIR-seq
Fastq reads were demultiplexed to single cells based on the CEL-Seq barcode located in positions 7-12 of R1.
R2 reads were filtered to contain the GFP sequence preceding the MIB (positions 1-37 of R2; GFP sequence: TGGGATTACACATGGCATGGATGAACTATACAAATAA, for ∆sseJ the sequence is TGGGATCACACATGGCATGGATGAACTATACAAATAA due to mutation in the plasmid during cloning). To account for sequencing errors, 1 mismatch was allowed for the GFP sequence identification.
The sequence of 10 nucleotides following the GFP (positions 38-47 of R2) is the bacterial barcode, i.e., the MIB sequence. We counted the number of reads that correspond to each bacterial barcode allowing 1 mismatch in the MIB sequence
Using this procedure, we generated a matrix with the counts of each mutant (MIB) in the pooled sample
Supplementary files format and content: Text files include MIB counts  for each mutant in the pooled sample

source_name: LB-grown cultures

growth_protocol: 25 S.Tm cultures of WT and 24 tagged SPI-2 effector mutants were grown in LB media at 37°C shaken for 16 hours.

extract_protocol:
RNA was extracted using RNeasy mini kit (Qiagen) with DNase-I treatment (45 min at 23°C). Resuspended RNA samples were treated with Turbo DNase (30 min at 37°C) for complete removal of bacterial DNA
scPAIR-seq relies on the CEL-seq library preparation protocol (Hashimshony et al., 2016) up to in-vitro transcription (IVT) step. After IVT, amplified RNA (aRNA) was treated with EXO-SAP enzyme (15 min at 37°C) to remove unused primers leftovers. aRNA sample was split and half of the sample volume (11 µl) was used to perform nucleic acid purification with RNAClean XP beads. The purified aRNA was converted to cDNA using superscript Ⅲ with specific GFP primer and cDNA was purified with AMPure XP beads. MIB library was amplified by 25 PCR cycles using a nested GFP primer to improve specificity of MIB amplification. To ensure exclusive sequencing of MIB reads by a custom GFP primer in the downstream steps, the nested GFP-primer was attached with a 5′-tail containing Illumina 3′-adaptor that was modified in its binding site for the Illumina sequencing primer

treatment_protocol: Cultures were normalized to the same OD600 value and equal volume was taken from all cultures to generate a pooled library of SPI-2 effector mutants. Pelleted pool was re-suspended with RLT buffer and stored at -80°C

tag: sample type

value: WT and 24 tagged SPI-2 effector mutants


title: Day 10 + Diff. medium rep2 pat. 1

description:
d10.+.diff_rep2_pat1
OC.J10.+.diff.rep2.pat.1_H3K27ac


title: primary human epididymis epithelial cells, donor 2, scRNAseq

description: 10x Genomics

value: male

value: normal


title: DELTA-5 HA 24h rep1

city: Montpellier

first: Amal

last: Makrini

address: 1919 route de mende

institute: IGMM

data_processing:
All sequencing reads were aligned to the hg19 genome using Bowtie2 or TopHat2 (allowing 2 mismatches, keeping uniquely aligned reads only).
For RNA-seq experiments, reads that aligned to + or – strand were seperated and processed seperately to produce wig files with strand specific enrichment scores.
For ChIP-seq experiments, replicates were merged and all wig files were rescaled to normalize the enrichment scores to reads per million.
Peak-callings were performed using parameters from the Thresholding function of IGB (thresh: background level, min run: minimum bin to be considered as enriched, min gap: minimum gap between two peaks to be considered as distincts) fed to an in-house script.
For HiC data, raw sequencing reads were cleaned using by default parameters of HiCUP pipeline and HOMER pipeline was then used to generate contact maps and PCA.
Assembly: hg19
Supplementary files format and content: wig

source_name: Raji B cells

growth_protocol: Human Raji B cells were cultured under standard growth conditions and transfected with full-length recombinant Rpb1 expression plasmids (rWT, YFFF, and Δ5).

extract_protocol:
Cells were harvested after 24 or 48 hours of α-amanitin treatment to perform western-blot analysis. Since Δ5 is unstable after 48 hours of α-amanitin treatment, cells were harvest after 24 hours to perform ChIP using specific antibodies (HA, H3K4me1, H3K4me3 and CTCF) or extraction of Total RNA, PolyA-RNA and chromatin-associated RNA (ChrRNA) using corresponding protocols as described in manuscript.
ChIP-seq libraries were prepared with Illumina's ChIP Sample Prep Kit according to standard protocol. RNA-seq libraries were prepared with Illumina's Small RNA Library Prep Kit by using a modified protocol described in manuscript. HiC libraries were prepared by proceeding to end-repair, A-tailing followed by the ligation of Illumina adaptaters and amplification.

treatment_protocol: Polyclonal cell lines were established after G418 (1 mg/mL) selection for 2-3 weeks. Expression of mutant Rpb1 was induced by culturing the cells in Tetracycline free medium. 24 hours after inductioncells were cultured in the presence of 2 µg/ml of α-amanitin to inhibit endogenous Pol II.

value: B lymphocyte

value: Burkitt's lymphoma

value: Raji

tag: antibody

value: HA (Abcam Reference:ab9110 and lot:913392)

tag: time


title: Rec-YF3 ChrRNA 24h rep2

description:
Rec-YF3_ChrRNA_24h_Merge_rep1_2_neg.wig
Rec-YF3_ChrRNA_24h_Merge_rep1_2_pos.wig

tag: strain

tag: molecule subtype

value: chromatin-associated RNA (ChrRNA)


title: DELTA-5 HA 24h rep2

data_processing:
All sequencing reads were aligned to the hg19 genome using Bowtie2 or TopHat2 (allowing 2 mismatches, keeping uniquely aligned reads only). The data Rec-WT HA 24h rep2, DELTA-5 HA 24h rep2, Rec-WT Input 24h rep1, Delta5 Input 24h rep1DELTA-5 H3K4me1 24h rep1, Rec-WT H3K4me1 24h rep1, DELTA-5 H3K4me3 24h rep1, Rec-WT H3K4me3 24h rep1, Rec-WT HA 24h rep1, DELTA-5 HA 24h rep1, DELTA-5 CTCF 24h rep1 & rep2 and Rec-WT CTCF 24h rep1 & rep2  were spiked using drosophila S2 cells but the spikeIn were not used during the bioinformatic processing.
For RNA-seq experiments, reads that aligned to + or – strand were seperated and processed seperately to produce wig files with strand specific enrichment scores.
For ChIP-seq experiments, replicates were merged and all wig files were rescaled to normalize the enrichment scores to reads per million.
Peak-callings were performed using parameters from the Thresholding function of IGB (thresh: background level, min run: minimum bin to be considered as enriched, min gap: minimum gap between two peaks to be considered as distincts) fed to an in-house script.
For HiC data, raw sequencing reads were cleaned using by default parameters of HiCUP pipeline and HOMER pipeline was then used to generate contact maps and PCA.
Assembly: hg19
Supplementary files format and content: Wig files

extract_protocol:
Cells were harvested after 24 or 48 hours of α-amanitin treatment to perform western-blot analysis. Since Δ5 is unstable after 48 hours of α-amanitin treatment, cells were harvest after 24 hours to perform ChIP using specific antibodies (HA, H3K4me1, H3K4me3 and CTCF) or extraction of Total RNA, PolyA-RNA and chromatin-associated RNA (ChrRNA) using corresponding protocols as described in manuscript. Drosophila cells were added as spike-in for ChIP experiments but not used for normalisation.
ChIP-seq libraries were prepared with Illumina's ChIP Sample Prep Kit according to standard protocol. RNA-seq libraries were prepared with Illumina's Small RNA Library Prep Kit by using a modified protocol described in manuscript. HiC libraries were prepared by proceeding to end-repair, A-tailing followed by the ligation of Illumina adaptaters and amplification.

tag: spike-in

value: Drosophila S2 cells


title: Rec-WT Input 24h rep1

value: Rec-WT


title: Delta5 Input 24h rep1


title: Rec-WT HA 24h rep2


title: DELTA-5 ChrRNA 24h rep1

description:
DELTA-5_ChrRNA_24h_Merge_rep1_2_neg.wig
DELTA-5_ChrRNA_24h_Merge_rep1_2_pos.wig


title: Rec-WT ChrRNA 24h rep2

description:
Rec-WT_ChrRNA_24h_Merge_rep1_2_neg.wig
Rec-WT_ChrRNA_24h_Merge_rep1_2_pos.wig


title: Rec-YF3 ChrRNA 24h rep1


title: PESA Study participant mRNA  SUAZ_074_v1

source_name: WBS

tag: tissue

value: WBS

value: Male

tag: age

value: Focal


title: P15-R2-D7

city: Iowa City

first: Andrew

middle: P

last: Voigt

state: IA

address: 375 Newton Road

institute: University of Iowa

data_processing:
FASTQ files were generated from the raw BCL files using Illumina’s bcl2fastq conversion program.
Reads were mapped to the human genome hg19 with STAR (version = 020201) and quantified with the featureCounts function from the Rsubread package (version = 1.28.1).
Differential expression analysis between the best producers (top 4 lines) vs the worst producers (bottom four lines) with edgeR. Technical replicates were corrected with voom::duplicateCorrelation().
Genome_build: hg19
Supplementary_files_format_and_content: Raw counts, logCPMs, and full differential expression results are provided in supplemental spreadsheets

extract_protocol:
Human induced pluripotent stem cell (hiPSC) samples were collected at two time points for RNA sequencing (day zero and seven, each time point in triplicate). Cell pellets were collected and flash frozen. Total RNA was extracted by a phenol/chloroform method using TRIzol LS reagent (ThermoFisher Scientific, MA) according to the manufacturer's guidelines.
RNeasy kit (Qiagen, Valencia, CA), Illumina TruSeq RNA Sample Preparation v2 library preparation kit (Illumina, San Diego, CA)

tag: timepoint

value: hiPSC-derived retinal organoids


title: P15-R3-D7


title: Rec-WT HA 24h rep1

tag: genotype


title: P15-R1-D7


title: PESA Study participant mRNA  SUAZ_080_v1

tag: gender

tag: pesa score

value: Intermediate


title: Rec-WT CTCF 24h rep2

tag: cell type

value: CTCF(Diagenode Reference:C15410210-50 and lot:A2359-00234P)


title: P13-R2-D7


title: P14-R3-D7


title: P13-R3-D7


title: P12-R3-D7


title: P14-R1-D7


title: P13-R1-D7


title: P12-R2-D7


title: P12-R1-D7


title: Rec-WT ChrRNA 24h rep1


title: P11-R3-D7


title: P11-R2-D7


title: DELTA-5 CTCF 24h rep2


title: P10-R3-D7


title: P11-R1-D7


title: P10-R2-D7


title: P10-R1-D7


title: DELTA-5 ChrRNA 24h rep2


title: P9-R3-D7


title: P14-R2-D7


title: P8-R1-D7


title: P7-R3-D7


title: P8-R2-D7


title: P7-R2-D7


title: P8-R3-D7


title: P7-R1-D7


title: P9-R1-D7


title: P6-R3-D7


title: P5-R1-D7


title: P5-R2-D7


title: P4-R3-D7


title: P6-R1-D7


title: P4-R2-D7


title: P5-R3-D7


title: P4-R1-D7


title: P3-R3-D7


title: P3-R2-D7


title: P2-R2-D7


title: P2-R3-D7


title: P3-R1-D7


title: P1-R2-D7


title: P2-R1-D7


title: P15-R3-D0


title: P1-R1-D7


title: P15-R2-D0


title: P9-R2-D7


title: P6-R2-D7


title: PESA Study participant mRNA  SUAZ_052_v1


title: P14-R3-D0


title: P13-R3-D0


title: P13-R2-D0


title: P15-R1-D0


title: P12-R2-D0


title: P11-R2-D0


title: P10-R3-D0


title: P11-R3-D0


title: P10-R1-D0


title: P12-R1-D0


title: P9-R3-D0


title: P9-R2-D0


title: P10-R2-D0


title: P1-R3-D7


title: P8-R2-D0


title: P8-R3-D0


title: P9-R1-D0


title: P7-R3-D0


title: P7-R1-D0


title: P7-R2-D0


title: P11-R1-D0


title: P6-R3-D0


title: P14-R2-D0


title: P14-R1-D0


title: P6-R2-D0


title: P12-R3-D0


title: P5-R2-D0


title: P5-R1-D0


title: P4-R2-D0


title: P13-R1-D0


title: P8-R1-D0


title: P6-R1-D0


title: P4-R3-D0


title: P4-R1-D0


title: P3-R2-D0


title: P2-R2-D0


title: P2-R3-D0


title: P2-R1-D0


title: P1-R1-D0


title: P3-R1-D0


title: P1-R3-D0


title: P5-R3-D0


title: P29_EB300, replicate 1, scRNAseq

city: Urbana

first: CheMyong

middle: Jay

last: Ko

state: Illinois

address: 2001 S. Lincoln Ave.

department: Comparative Biosciences

institute: University of Illinois at Urbana Champaign

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v3.0.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) using a custom reference made for genome Rnor6.0 and Ensembl 96 gene models following 10X's method for the pre-built mouse reference
Assembly: Ensembl Rnor6.0, version 96
Supplementary files format and content: Tab-separated values files and matrix files

organism: Rattus norvegicus

source_name: Hypothalamus

extract_protocol:
After collagenase-assisted tissue dissociation, single-cell suspensions were converted into individually barcoded cDNA libraries with the Single-Cell 3’ Chromium kit version 3 from 10X Genomics.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Mixed

value: Hypothalamus

value: Sprague Dawley

value: 4 weeks old

tag: Sex

value: female


title: Original E21.5 Healthy Control

first: Peter

last: Bazeley

address: 9500 Euclid Avenue

department: Lerner Research Institute

institute: Cleveland Clinic

data_processing:
BCL files from the Novaseq were converted to FastQ files with the Cell Ranger (10x Genomics Cell Ranger 4.0.0) mkfastq function. An alignment reference was constructed using the Cell Ranger mkref function applied to the Ensembl Rattus norvegicus genome fasta (http://ftp.ensembl.org/pub/release-104/fasta/rattus_norvegicus/dna/Rattus_norvegicus.Rnor_6.0.dna.toplevel.fa.gz) and GTF (http://ftp.ensembl.org/pub/release-101/gtf/rattus_norvegicus/Rattus_norvegicus.Rnor_6.0.101.gtf.gz, filtered with the Cell Ranger mkgtf function with option “—attribute=gene_biotype:protein_coding”). Reads were aligned and genes quantified with the Cell Ranger count function.
Downstream analysis was performed with R (version 4.1.0.) and Seurat (version 4.0.3). Default options for each function were used unless otherwise specified. Each group of samples was analyzed separately. Samples were filtered to exclude cells with > 20% mitochondrial expression, = 100 using bcftools (version 1.12) and cell doublet assignments were calculated with Variational Inference for Reconstructing Ensemble Origins (Vireo, version 0.5.6). UMI counts were normalized with Seurat function SCTransform, using method “qpoisson”, and additionally corrected for percent mitochondrial expression.
The 2 healthy control samples were integrated with functions SelectIntegrationFeatures (using and returning the top 3000 most variable genes), PrepSCTIntegration, FindIntegrationAnchors (using normalization method “SCT” and 5 anchors), and IntegrateData (using normalization method “SCT”). Principal component analysis (PCA) was performed on the integrated counts with function RunPCA. Clusters were identified using functions FindNeighbors (using the first 30 PCs) and FindClusters (using a range of resolutions from 0 to 1.0 at every 0.05 increment). Marker genes conserved across the 2 healthy control samples were identified for each cluster with function FindConservedMarkers and annotated with R package AnnotationHub (version 1.54.1). Marker gene lists were manually analyzed to determine the cell type of each cluster. For the nitrofen control and nitrofen CDH samples, PCA was performed on the SCT-normalized counts; clustering and marker analysis was performed for each sample separately, using a resolution of 1.0 with FindClusters. Uniform Manifold Approximation and Projection (UMAP) was applied to the first 30 principal components (PCs) with function RunUMAP.
Assembly: Rnor_6.0
Supplementary files format and content: tab-separated, comma-separated, and matrix files.

source_name: lung

extract_protocol:
Pooled NC and CDH specimens combined fetuses from two separate pregnant dams, and the two sets of pooled 2HC specimens each came from a different mother. Pooled lungs were digested into a single cell suspension, as previously described 46. Briefly, lung tissue was minced and dissociated in 0.1% collagenase A (Sigma Aldrich), 0.04% DNase I (Sigma Aldrich), and 0.5 mM CaCl2 in 3 ml of 10 mg/mL Dispase II (Sigma Aldrich. Incubations were performed on an orbital shaker at 37°C for a total of 45 min. Cell clumps were disrupted via pipetting, and the suspension was passed through a 40-micron cell strainer to remove any undigested tissue pieces or aggregates. Pelleted cells were then resuspended in 1% BSA in PBS to prevent   aggregation. Cell viability and concentration were assessed using propidium iodide and acridine orange with a CellDrop automated cell counter (DeNovix, Wilmington, DE).
A quantity of 10,000 single cells were targeted using the 10x Genomics Chromium Controller (Pleasanton, CA) for cDNA synthesis and barcoding. cDNA quality and quantity of each sample were assessed using a Bioanalyzer High Sensitivity DNA assay, and samples were constructed following the 10x Genomics Chromium Single Cell 3’ V3 protocol (CG000183 Rev A). Sample libraries were then assessed for proper construction using the same Bioanalyzer assay. Samples were pooled equally, and the resulting DNA library was quantified using the sparQ Universal Library Quantification kit (Quantabio, Beverly, Massachusetts) to inform sample preparation for downstream Illumina sequencing. Libraries were denatured and sequenced on   an Illumina Novaseq 6000 (San Diego, CA) high-throughput sequencing platform with 28 cycles for the forward read and 91 cycles for the reverse read. Each cell was sequenced to a minimum of 20,000 reads per cell per manufacturer’s recommendations. We achieved an average sequencing saturation of 33.1%.

treatment_protocol: Cleveland Clinic Institutional Animal Care and Use Committee (IACUC) approval was obtained (Protocol #2019-2282), and experiments were performed in accordance with all relevant guidelines and regulations for the care and use of laboratory animals. This study was carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Time-mated, pregnant Sprague Dawley rats (Envigo, Hackensack, NJ) with a mean ± SD weight of 230.3 ± 5.2 g were gavage fed 100 mg nitrofen (2,4-diclorophenyl p-nitrophenyl-ether; Sigma Aldrich, St. Louis, MO) dissolved in olive oil on embryonic day 9.5 (E9.5) to induce fetal diaphragmatic hernia, pulmonary hypoplasia and pulmonary hypertension that is similar to human disease. 1 mL olive oil alone was gavage fed to healthy controls. Pregnant dams were euthanized with CO2 asphyxiation and subsequent cervical dislocation. Fetuses were immediately harvested by cesarean section on E21.5 and decapitated prior to proceeding with dissection to examine the diaphragm and isolate the left lung. Only fetuses with left-sided diaphragmatic defects >50% of the area of the diaphragm were selected for analysis in the CDH group. We achieved on average 73% of fetuses with left-sided diaphragmatic defects. Lungs from nitrofen exposed fetuses without a diaphragmatic defect were analyzed as a second control population in order to distinguish the effect of nitrofen from that of lung compression. Left lungs (those ipsilateral to diaphragmatic defect in the CDH group) from all three experimental groups were carefully dissected free of all surrounding structures (including the heart, aorta, esophagus, trachea, and right lung) in cold PBS using 2.5x loupe magnification. Lungs were phenotyped by measuring weight, length and width. For control lungs, the largest lungs   were selected for scRNA-seq. For the CDH group, we selected left lungs that were smallest in relation to the matched right lung in order to choose ones that were most impacted by left lung compression. There were fewer NC lungs to select from, but the largest ones were used for scRNA-seq. Ratios of left-to-right lung length were calculated from photographs.

value: ling

value: Sprague-Dawley

tag: growth stage

value: fetus

value: embryonic day 21.5

value: control

tag: disease

value: no


title: P29_intact, replicate 1, scRNAseq

description: 10x Genomics


title: E21.5 Nitrofen CDH

description: 10x Genomics

value: lung

tag: treatment

value: nitrofen

value: yes


title: New E21.5 Healthy Control

description: 10x Genomics


title: E21.5 Nitrofen Control

description: 10x Genomics


title: Male-ipsi-1

city: Marseille

middle: J

last: Saurin

address: Campus de Luminy

institute: IBDM / CNRS

description:
Counts_all.txt
DESeq2_DGE_all.txt

data_processing:
Image analysis and base calling were performed using RTA 2.7.7 and bcl2fastq 2.20.0.422.
Reads were mapped to the mm10 genome using subread-align (Subread package v2.0.3) on the command line using default parameters
Reads aligning t0 mm10  exons were counted using featureCounts (Subread package v2.0.3) on the command line using default paramters
Differentially expressed genes were called using DESeq2 (version 1.3.0)
Assembly: mm10
Supplementary files format and content: Exon counts from Subread featureCounts in Miscosoft Excel format
Supplementary files format and content: Exon counts from Subread featureCounts in tab-delimited plain text format
Supplementary files format and content: Differentially expressed genes from DESeq2 analyses in tab-delimited plain text format

source_name: spinal cord tissue from the dorsal horn region ipsilateral to the formalin-injected hind paw

extract_protocol:
Dorsal horns of the spinal cord were rapidly dissected and immediately frozen on dry ice. Samples were stored at -80°C untill RNA extraction. Total RNA was extracted using Quiagen Rneasy extraction kit, according to manufacturer's recommendations.
P2736 : Full length cDNA were generated from 4ng of total RNA  using SMART-SeqX v4 UltraX Low Input RNA Kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 10 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundreds pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, USA) followed by 12 cycles of library amplification. Following purification with SPRIselect beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoreris.

treatment_protocol: A 2% solution of formalin (diluted in PBS) was injected in mice's hidpaw. Mice nocifensive behavior was evaluated during the first hour post-injection. Two hours following formalin administration, mice were deeply anesthesized and dorsal horn of the spinal cord was dissected.

tag: formalin

value: 2H post formalin injection

value: male


title: Male-ipsi-5

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Male-ipsi-6

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Male-ipsi-3

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Male-ipsi-4

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F2/Metestrus-ipsi-4

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F2/Metestrus-ipsi-1

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F2/Metestrus-ipsi-2

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F2/Metestrus-ipsi-5

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: P1-R2-D0

value: hiPSC-derived retinal organoids


title: Male-ipsi-2

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F2/Metestrus-ipsi-3

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: P3-R3-D0


title: F1/Proestrus-ipsi-6

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Neutrophils_Trt24hr-1 [Sample4]

city: Newton

first: Matt

last: Kanke

state: MA - Massachusetts

address: 112 Harding Street

institute: Amgen

data_processing:
RNA sequencing reads (Illumina HiSeq platform, 75 bp paired end sequencing) were aligned to Human genome build 38 and Fragments per Kilobase per Million sequenced, Quantile normalized (FPKQ) values were determined using Array Suite software (Omicsoft, Cary, NC) and in-house software.
Assembly: The Salmon v1.4.0 [43] was used with default parameters to quasi-map RNA-seq reads to the target transcriptome (Gencode vM27) for quantification. The count files were then imported into DESeq2 (v1.34.0 in R4.1) [44] using tximport and DESeqDataSetFromTximport [45] packages. A minimal pre-filtering cutoff of 10 reads total across samples for each gene was done to remove lowly expressed genes.
Supplementary files format and content: csv files with normalized counts for each time point

source_name: BM

growth_protocol: Specified in experimental design

extract_protocol: Each of the cell type was either untreated or treated with 500 ng/ml of rmIL-36γ (R&D Technologies) for 6h or 24h and RNA from extracted using RNeasy isolation kit (Qiagen) according to manufacturer’s guidelines. Depending on the RNA amount available, 0.7- 5.0 ng of total RNA was used as the starting input to generate the full-length cDNA by using the SMART-Seq v4 Ultra-low Input RNA Kit for Sequencing (Takara Catalog No.634890). RNA was primed by the 3′ SMART-Seq CDS Primer II A with the SMART-Seq v4 Oligonucleotide for template switching at the 5′ end of the transcript during the first-strand cDNA synthesis. Preparation of SMARTer cDNA was sequentially amplified by following the LD-PCR procedure, which was carried out via the thermal cyclic program according to the input amount of total RNA: 11 PCR cycles for 1.0-5.0ng RNA samples and 15 PCR cycles for 0.7-1.0 ng RNA samples. The LD-PCR amplified cDNA was purified and further validated in the Agilent High Sensitivity D5000 ScreenTape (Agilent, Catalog No.5067-5592) on the Agilent 4200 TapeStation. Then, 300pg cDNA products were used for library preparation in each reaction by using the Illumina Nextera XT kit and following the manual instruction (Illumina, Catalog No.FC-131-1096). The products of low-input cDNA libraries were then barcoded after by 12 cycles of thermal program with specific index adapters for Illumina sequencing (Nextera XT Index Set D, Illumina, Catalog No. FC-131-2004). The resulting libraries were cleaned up using 0.8x AMPure XP beads (Beckman Coulter, Catalog No. A63881) and eluted in 25 µl of resuspension buffer. After library QC and quantitation, libraries were sequenced to a minimum depth of 45 million paired-end/Dual-indexed 2x150bp reads on an Illumina HiSeq4000).

treatment_protocol: Specified in experimental design

value: Neutrophils

value: BM

value: WT

value: Treated - recombinant mouse IL-36g


title: F1/Proestrus-ipsi-4

description:
Counts_all.txt
DESeq2_DGE_all.txt

value: female


title: Neutrophils_UT24hr-3 [Sample3]

value: Untreated


title: F1/Proestrus-ipsi-2

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F1/Proestrus-ipsi-3

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: F1/Proestrus-ipsi-1

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Neutrophils_Trt6hr-3 [Sample6]


title: F2/Metestrus-ipsi-6

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: Neutrophils_Trt6hr-2 [Sample5]


title: Neutrophils_Trt24hr-3 [Sample6]


title: Neutrophils_Trt24hr-2 [Sample5]


title: Neutrophils_UT24hr-2 [Sample2]


title: NK_Trt24hr-2 [Sample5]

source_name: Spleen

value: NK

value: Spleen


title: Neutrophils_UT6hr-2 [Sample2]


title: NK_Trt24hr-1 [Sample4]


title: Neutrophils_UT24hr-1 [Sample1]


title: Neutrophils_Trt6hr-1 [Sample4]


title: F1/Proestrus-ipsi-5

description:
Counts_all.txt
DESeq2_DGE_all.txt


title: NK_Trt24hr-3 [Sample6]


title: NK_UT24hr-3 [Sample3]


title: NK_Trt6hr-3 [Sample7]


title: NK_UT24hr-2 [Sample2]


title: NK_UT24hr-1 [Sample1]


title: NK_Trt6hr-2 [Sample6]


title: NK_UT6hr-4 [Sample4]


title: NK_Trt6hr-1 [Sample5]


title: NK_UT6hr-2 [Sample2]


title: Neutrophils_UT6hr-3 [Sample3]


title: Mono_Trt24hr-3 [Sample6]

value: Mono


title: Mono_Trt24hr-1 [Sample4]


title: Mono_Trt24hr-2 [Sample5]


title: NK_UT6hr-3 [Sample3]


title: Mono_UT24hr-1 [Sample1]


title: NK_UT6hr-1 [Sample1]


title: Mono_Trt6hr-3 [Sample6]


title: Neutrophils_UT6hr-1 [Sample1]


title: Mono_Trt6hr-1 [Sample4]


title: Mono_UT24hr-2 [Sample2]


title: Mono_UT6hr-3 [Sample3]


title: Mono_UT6hr-1 [Sample1]


title: DC2_Trt24hr-3 [Sample6]


title: DC2_Trt24hr-2 [Sample5]


title: DC2_UT24hr-2 [Sample2]


title: DC2_UT24hr-1 [Sample1]


title: DC2_Trt6hr-3 [Sample6]


title: DC2_UT24hr-3 [Sample3]


title: DC2_Trt6hr-2 [Sample5]


title: DC2_Trt6hr-1 [Sample4]


title: Mono_UT24hr-3 [Sample3]


title: DC2_UT6hr-2 [Sample2]


title: Mono_Trt6hr-2 [Sample5]


title: DC1_Trt24hr-2 [Sample5]


title: DC1_Trt24hr-1 [Sample4]


title: DC2_UT6hr-1 [Sample1]


title: DC1_Trt24hr-3 [Sample6]


title: DC1_UT24hr-1 [Sample1]


title: DC1_UT24hr-3 [Sample3]


title: DC1_Trt6hr-3 [Sample6]


title: Mono_UT6hr-2 [Sample2]


title: DC2_Trt24hr-1 [Sample4]


title: DC1_UT6hr-3 [Sample3]


title: DC1_UT6hr-2 [Sample2]


title: BMDM_Trt24hr-3 [Sample6]

value: BMDM


title: DC1_UT6hr-1 [Sample1]


title: DC1_Trt6hr-2 [Sample5]


title: DC1_Trt6hr-1 [Sample4]


title: BMDM_Trt24hr-1 [Sample4]


title: DC2_UT6hr-3 [Sample3]


title: BMDM_UT24hr-2 [Sample2]


title: BMDM_Trt6hr-3 [Sample6]


title: BMDM_UT24hr-1 [Sample1]


title: BMDM_UT24hr-3 [Sample3]


title: BMDM_Trt6hr-1 [Sample4]


title: BMDM_Trt6hr-2 [Sample5]


title: BMDM_UT6hr-3 [Sample3]


title: BMDM_UT6hr-2 [Sample2]


title: BMDM_UT6hr-1 [Sample1]


title: DC1_UT24hr-2 [Sample2]


title: lung, SARS2, WT, mouse 7

city: New. York

first: Katrina

last: Mar

address: 417 E 68th St

institute: MSKCC

data_processing:
Raw reads of fastq format were first processed through in-house perl scripts. In this step, clean reads were obtained by removing reads containing adapter and low-quality reads from raw data. Index of the reference genome was built and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene and FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis was performed using the DESeq2 R package (1.20.0). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected P value less than 0.05 were considered significantly enriched by differential expressed genes.
Assembly: mm10
Supplementary files format and content: gene_count.txt: Normalized gene counts.
Supplementary files format and content: *_deg.txt: Differentially expressed genes (generated from DESeq2).
Supplementary files format and content: WT_SARS2vsKO_SARS2.all_GOenrich_significant.txt: Gene ontology (generated from clusterProfiler).

source_name: whole lung

extract_protocol:
Whole lungs were isolated from SARS-CoV-2 infected mice and homogenized in 600 µL TRIzol (Sigma-Aldrich, MA) with 3.2 mm stainless steel beads (NextAdvance, NY) in a Bullet Blender Storm Pro (NextAdvance, NY). Lung homogenates were clarified of debris by centrifugation at 800 xg for 5 minutes, diluted into fresh TRIzol (Sigma-Aldrich, MA) at a 1 to 9 ratio, and frozen at -80°C. For RNA isolation, chloroform was added to thawed samples at a 1 to 5 ratio, shaken for 15 seconds, then centrifuged for 15 minutes at 12,000 xg at 4°C. The aqueous phase was transferred to a fresh tube, mixed with 1 equal volume of 70% ethanol, then transferred to a RNeasy Mini Kit filter column (Qiagen, MD). Samples were processed per manufacturer’s instructions with on column DNase I treatment (Qiagen, MD).
Purified RNA was submitted to Novogene (CA) in DNA/RNA shield (Zymo Research, CA) for library preparation and RNA-Seq analysis. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dTTP for generation of a non-directional library. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. The clustering of the index-coded samples was performed according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.

value: whole lung


title: lung, SARS2, WT, mouse 6


title: BMDM_Trt24hr-2 [Sample5]


title: lung, SARS2, KO, mouse 7

value: Ly6e-del/del; Sox2-cre+


title: lung, SARS2, WT, mouse 3


title: lung, SARS2, KO, mouse 6


title: lung, SARS2, WT, mouse 5


title: lung, SARS2, KO, mouse 4


title: lung, SARS2, WT, mouse 4


title: lung, SARS2, KO, mouse 5


title: lung, SARS2, KO, mouse 1


title: lung, SARS2, WT, mouse 2


title: lung, SARS2, KO, mouse 3


title: lung, mock, WT, mouse 6

value: uninfected


title: lung, mock, WT, mouse 5


title: lung, mock, WT, mouse 4


title: lung, SARS2, KO, mouse 2


title: lung, mock, KO, mouse 2


title: lung, mock, WT, mouse 1


title: lung, mock, KO, mouse 1


title: lung, mock, WT, mouse 3


title: lung, mock, WT, mouse 2


title: lung, mock, KO, mouse 3


title: lung, mock, KO, mouse 4


title: eS27-FLAG, total fraction ribosome-protected fragment, biol rep 3 [L3_2c4totrfp]

city: Palo Alto

first: Maria

last: Barna

address: 240 Pasteur Dr, BMI 4500

data_processing:
library strategy: ribosome profiling
cutadapt version 2.4 (Martin, 2011) was used to trim 3’ adapter sequences from Read 1 with parameters “-j 0 -u 3 -a AGATCGGAAGAGCACAGTCTGAACTCCAGTCAC --discard-untrimmed -m 15”. In-line barcodes were demultiplexed using fastx_barcode_splitter.pl (http://hannonlab.cshl.edu/fastx_toolkit/) with parameters “--eol”. Unique molecular identifiers and in-line barcodes were extracted using umi_tools version 1.0.1 (Smith et al., 2017) with parameters “extract --extract-method=string --bc-pattern=NNNNNCCCCC --3prime”. Reads were filtered by quality using fastq_quality_filter (http://hannonlab.cshl.edu/fastx_toolkit/) with parameters “-Q33 -q 20 -p 70 -z”. To remove reads originating from rRNA, transfer RNA (tRNA), and small nuclear RNA (snRNA), reads aligning to these sequences using bowtie2 version 2.3.4.3 (Langmead & Salzberg, 2012) with parameters “-L 18” were discarded. Remaining reads were aligned using bowtie2 with parameters “--norc -L 18” to a reference GRCm38/mm10 mouse transcriptome that was derived from UCSC/GENCODE VM20 knownCanonical annotations filtered for transcripts associated with at least one of the following: a Uniprot ID, a RefSeq ID, or an Entrez ID. PCR duplicates were removed using umi_tools. RPFs were parsed for uniquely aligned reads and grouped by read length. Ribosome A site positions were determined by offsetting the distance of the 5’ end of each read to canonical start sites in each length group and adding 4 nucleotides. RPF reads aligning to the coding sequence (CDS) of a transcript (excluding the first 15 codons and last 5 codons of each CDS) were counted, using the above transcriptome annotation.
Assembly: mm10
Supplementary files format and content: reads_cds_v_samp.txt: Tab-delimited matrix of read counts within the coding sequences (excluding the first 15 and last 5 codons) of transcripts (rows) for all samples (columns)

growth_protocol: E14Tg2a.4 mESCs (Smith and Hooper, 1987) were a gift from Thom Saunder’s lab (University of Michigan). Cells were cultured in Knockout DMEM (ThermoFisher 10829-018) supplemented to a final concentration of 15% ES-qualified fetal bovine serum (MilliporeSigma ES-009-B, Burlington, MA), 1% non-essential amino acids (MilliporeSigma TMS-001-C), 2 mM L-glutamine (MilliporeSigma TMS-002-C), 1% penicillin/streptomycin (ThermoFisher 15140-122), 55 uM beta-mercaptoethanol (ThermoFisher 21985-023), and 1000 U/mL mouse leukemia inhibitory factor (mLIF, Gemini 400-495 10^7, West Sacramento, CA). mESCs were not routinely tested for mycoplasma contamination. Cell line identity was confirmed daily by observing colony morphology. Media was changed daily and cells were passaged every two days. To passage, plates with ~70% confluent colonies of mESCs were washed in Dulbecco′s Phosphate Buffered Saline (DPBS) (ThermoFisher 14190-250) and trypsinized (0.05% trypsin, dilution of ThermoFisher 15400-054 in DPBS) for 5 minutes at 37 C. Trypsin was neutralized by a double volume of media. Cells were immediately dissociated by vigorous pipetting, pelleted at 200 g for 3 minutes at room temperature, and resuspended in fresh warmed media for plating. Fresh plates were pre-coated at 37 C overnight with 0.1% gelatin (MilliporeSigma ES-006-B), which was aspirated prior to plating cells. Unless otherwise stated, cells were plated at a density equivalent to 5 x 106 cells per 10 cm plate.

extract_protocol:
Total RPFs and IP RPFs were extracted from TRIzol using the Direct-Zol Microprep Kit (Zymo R2060, Irvine, CA) according to the manufacturer protocol, omitting the DNAse digestion.
Ribosome profiling libraries were prepared following the published protocol of McGlincy and Ingolia (McGlincy & Ingolia, 2017) with modifications as stated below, using oligonucleotides synthesized by Integrated DNA Technologies (Coralville, IA) (Supplementary Table 7). After total and IP RPFs were extracted from TRIzol as described above, RPF volume was adjusted to 90 uL with Ultrapure water. 10 uL 3M NaOAc pH 5.5 (ThermoFisher AM9740) and 2 uL of 15 mg/mL GlycoBlue (ThermoFisher AM9515) were added before precipitating the RPFs in 150 uL 100% isopropanol overnight at -80 C. Precipitated RPFs were pelleted at 21,000 g for 30 minutes at 4 C, washed with ice-cold 80% ethanol in water, dried at room temperature for 10 minutes, and dissolved in Ultrapure water. RPFs were denatured at 80 C for 90 sec in denaturing sample loading buffer (McGlincy & Ingolia, 2017), then incubated on ice for 5 min before running on a 15% Tris-borate-EDTA-urea (TBE-urea) polyacrylamide gel. Fragments were size-selected using NI-800 and NI-801 (McGlincy & Ingolia, 2017) as 26-34 nt markers. Gel slices were freeze-thawed for 30 minutes at -80 C, crushed, and extracted at room temperature overnight in 400 uL RNA extraction buffer (McGlincy & Ingolia, 2017), then re-extracted with an additional 200 uL RNA extraction buffer. The combined 600 uL extraction was precipitated with 2 uL GlycoBlue and 750 uL 100% isopropanol overnight at -80 C. The RPFs were pelleted, washed, and dried as described above, then dissolved in 4 uL of 10 mM Tris pH 8, dephosphorylated, and ligated to barcoded linkers per the published protocol. Between replicates, barcodes were permuted among the samples. Unreacted linker was deadenylated and digested per the published protocol. The barcoded RPFs were pooled within each replicate and purified on a Zymo Oligo Clean & Concentrator column (Zymo D4060) according to manufacturer protocol. Pooled RPFs were diluted to 100 ng/uL and 1 ug was used as input for rRNA depletion using RiboZero Gold (part of Illumina 20020598, San Diego, CA) per manufacturer protocol. rRNA-depleted RPFs were purified on a Zymo Oligo Clean & Concentrator column, then reverse-transcribed per the published protocol. Template RNA was degraded by alkaline hydrolysis per the published protocol. cDNA was purified on a Zymo Oligo Clean & Concentrator column, denatured in denaturing sample loading buffer, and size-selected on a 10% TBE-urea gel, as marked by NI-800 and NI-801 that had been ligated and reverse-transcribed in parallel with the samples. Gel slices were freeze-thawed for 30 minutes at -80 C, crushed, and extracted at room temperature overnight in 400 uL DNA extraction buffer (McGlincy & Ingolia, 2017), then re-extracted with an additional 200 uL DNA extraction buffer. The combined 600 uL extraction was precipitated with 2 uL GlycoBlue and 750 uL 100% isopropanol overnight at -80 C. The cDNA was pelleted, washed, and dried as described above, then resuspended in 15 uL 10 mM Tris pH 8. cDNA was circularized by adding 2 uL 10X CircLigase I buffer, 1 uL of 1 mM ATP, 1 uL of 50 mM MnCl2, and 1 uL of CircLigase I (Lucigen CL4111K, Middleton, WI) to the 15 uL of cDNA and incubating at 60 C for 12 hours, then 80 C for 10 minutes. Circularized cDNA was purified on a RNA Clean & Concentrator column. cDNA concentration was quantified by qPCR per the published protocol except using SsoAdvanced Universal SYBR Green qPCR master mix (Biorad 1725274) and following manufacturer protocol. Library construction to add indexing primers was performed per the published protocol, using a different reverse primer for each replicate. PCR products were purified using a Zymo DNA Clean & Concentrator column (Zymo D4003). Size selection was performed on a 8% TBE-urea gel, with the lower bound marked by NI-803 (McGlincy & Ingolia, 2017) that had undergone library construction in parallel with the samples, and the upper bound at 170 nt as marked by O’Range 20 bp DNA ladder (Thermo SM1323). Gel slices were extracted as described above. DNA was precipitated as described above, except using 1.25 uL of 20 ug/mL glycogen (Themo 10814-010) instead of GlycoBlue in the precipitation and incubating at -80 C for 2 hours. The pellet was resuspended in 10 mM Tris pH 8. Library quality and concentration was analyzed on an Agilent 2100 Bioanalyzer (High-Sensitivity DNA) at the Stanford Protein and Nucleic Acid Facility. Libraries were sequenced by Novogene (Sacramento, CA) on an Illumina HiSeq 4000 with 150 bp reads.

treatment_protocol: IP lysis buffer: 25 mM Tris pH 7.5 (ThermoFisher AM9850G, AM9855G), 150 mM NaCl (ThermoFisher AM9760G), 15 mM MgCl2 (ThermoFisher AM9530G), 1% v/v Triton-X 100 (MilliporeSigma X100-500ML), 8% v/v glycerol (MilliporeSigma G6279), 1 mM DTT (MilliporeSigma 43815), 1X Complete Mini Protease Inhibitor EDTA-free (Roche 11836170001), 0.5% w/v deoxycholate (MilliporeSigma S1827), 200 ug/mL cycloheximide (MilliporeSigma C7698), 0.02 U/uL Turbo DNAse (ThermoFisher AM2239), 0.2 U/uL Superase RNAse Inhibitor (ThermoFisher AM2696) in Ultrapure distilled water (ThermoFisher 10977-015).    Sucrose cushion buffer: 25 mM Tris pH 7.5 (ThermoFisher AM9850G, AM9855G), 150 mM NaCl (ThermoFisher AM9760G), 15 mM MgCl2 (ThermoFisher AM9530G), 1 mM DTT (MilliporeSigma 43815), 1X Complete Mini Protease Inhibitor EDTA-free (Roche 11836170001), 200 ug/mL cycloheximide (MilliporeSigma C7698) in Ultrapure distilled water (ThermoFisher 10977-015).    Wash buffer 1: equivalent to IP lysis buffer but omitting glycerol, protease inhibitor, RNAse inhibitor, and DNAse    Wash buffer 2: equivalent to wash buffer 1, but with 300 mM NaCl    Western elution buffer: 4% v/v SDS (MilliporeSigma 436143), 125 mM Tris pH 7.0 (ThermoFisher AM9850G) in Ultrapure distilled water (ThermoFisher 10977-015).    CRISPR-edited mESCs and control lines were grown to ~50% confluence in 2x15 cm plates. Three biological replicates (clones) were included for each genotype. Cells were treated with 100 ug/mL cycloheximide (CHX) for 2 minutes at 37 C, then washed twice with 20 mL DPBS containing 100 ug/mL CHX. Cells were scraped into 5 mL DPBS+CHX and centrifuged at 200 g for 3 minutes at 4C, then washed again in DPBS+CHX. 800 uL IP lysis buffer without Superase RNAse Inhibitor was added to the pellet and vortexed for 30 seconds with 30 seconds rest for 3 cycles, then rotated at 4 C for 30 minutes. Lysate was clarified by centrifuging at 700 g for 5 min at 4C, then again at 7000 g for 5 min at 4C. RNA concentration was measured by Nanodrop 2000 (Thermo). By adding more IP lysis buffer without RNAse inhibitor, the clarified lysate was adjusted to a concentration of 1500 ng/uL in 600 uL total volume. 6 uL RNase A (Thermo EN0531) and 3.6 uL RNAse T1 (Thermo EN0541) were added, and the digestion reaction was rotated for 30 minutes at room temperature. RNAse digestion was quenched by placing it on ice and adding 30 uL of Superase RNAse Inhibitor. Two 250 uL aliquots of digested lysate were each layered over 750 uL of sucrose cushion buffer (see above). The cushions were ultracentrifuged in a TLA 120.2 rotor (Beckman, Indianapolis, IN) at 100,000 rpm for 1 hour at 4 C. Each pellet was rinsed once with 1 mL ice-cold Ultrapure water, then resuspended by pipetting and shaking in 250 uL IP lysis buffer containing Superase RNAse Inhibitor. An aliquot of the resuspended pellet was reserved in TRIzol (ThermoFisher 15596-018) as the total RPF fraction. The two resuspended pellets from each replicate were combined and added to 200 uL mouse IgG beads (MilliporeSigma A0919) which had been equilibrated twice for 5 min each at 4 C in an equal volume of IP lysis buffer with RNAse inhibitor. The samples were pre-cleared with the IgG beads for 1 hour at 4 C on a rotator, then transferred to 200 uL anti-FLAG beads (MilliporeSigma A2220), which had been equilibrated twice for 5 min each at 4 C in an equal volume of IP lysis buffer with RNAse inhibitor. The immunoprecipitation reaction was rotated for 2 hours at 4C, washed with 400 uL wash buffer 1 three times for 5 min each at 4C, and washed with 400 uL wash buffer 2 three times for 5 min each at 4 C. For ribosome profiling, an aliquot of the beads was incubated in TRIzol for 5 minutes at room temperature and reserved as the IP RPF fraction.

tag: cell line

value: mouse embryonic stem cells


title: WT, total fraction ribosome-protected fragment, biol rep 3 [L3_5a3totrfp]


title: eS27L-FLAG, IP fraction ribosome-protected fragment, biol rep 3 [L3_4b7iprfp]

value: IP fraction ribosome-protected fragment


title: eS27L-FLAG, total fraction ribosome-protected fragment, biol rep 3 [L3_4b7totrfp]


title: eS27-FLAG, IP fraction ribosome-protected fragment, biol rep 3 [L3_2c4iprfp]


title: WT, total fraction ribosome-protected fragment, biol rep 2 [L2_5a2totrfp]


title: eS27L-FLAG, total fraction ribosome-protected fragment, biol rep 2 [L2_4a3totrfp]


title: eS27-FLAG, total fraction ribosome-protected fragment, biol rep 2 [L2_2c3totrfp]


title: eS27L-FLAG, IP fraction ribosome-protected fragment, biol rep 2 [L2_4a3iprfp]


title: eS27-FLAG, IP fraction ribosome-protected fragment, biol rep 2 [L2_2c3iprfp]


title: lung, SARS2, WT, mouse 1


title: eS27L-FLAG, IP fraction ribosome-protected fragment, biol rep 1 [L1_4a2iprfp]


title: eS27L-FLAG, total fraction ribosome-protected fragment, biol rep 1 [L1_4a2totrfp]


title: WT, total fraction ribosome-protected fragment, biol rep 1 [L1_5a1totrfp]


title: eS27-FLAG, total fraction ribosome-protected fragment, biol rep 1 [L1_2b12totrfp]


title: eS27-FLAG, IP fraction ribosome-protected fragment, biol rep 1 [L1_2b12iprfp]