title: SedSeq~tg_5~L112~spombe_spikeIn~BY4741~30C~10min~none~BR2~Pellet

data_processing:
Sequencing reads were trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename F02 AD-JB-F02_S44_R1_001.fastq.gz AD-JB-F02_S44_R2_001.fastq.gz). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted --readFilesCommand gunzip -c  --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/F02/F02_ --readFilesIn trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz). The estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/F02 --fr-stranded --bootstrap-samples=50 -t 1 trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz).
Both NanoLuc spike-in RNA and S. pombe total RNA was added and reads were mapped to spike_saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled.fasta (STAR) using the annotation file spike_saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled_geneid.gff3, and spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding_Spombe_cds.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample

extract_protocol:
Biochemical fractionation was completed similarly to Wallace et al. [2015], with the major exception that 20,000 g for 10 min was used rather than the original 100,000 g for 20 min. In short, 50 mL cultures of treated yeast were harvested by centrifugation at 3000 g for 5 minutes, then resuspended in 100 ¬µL of soluble protein buffer (SPB: 20 mM HEPES, pH 7.4, 140 mM KCl, 2 mM EDTA, 0.1 mM TCEP, 1:200 protease inhibitor (Millipore #539136), 1:1000 SUPERase‚Ä¢In RNase Inhibitor (Invitrogen #AM2696)), and flash frozen in liquid nitrogen as a pellet in a 2 mL Eppendorf Safe-Lock tube (Eppendorf #0030123620) with a 7 mm steel ball (Retsch #05.368.0035). The cells were then lysed using a Retsch MM400 for 5x90s at 30 Hz, chilling in liquid nitrogen between each shaking repeat. The lysed cells were resuspended in 600 ¬µL of SPB, and 100 ¬µL of total sample was transferred to 300 ¬µL of Trizol LS (Invitrogen #10296010). The remainder was centrifuged for 30 seconds at 3000 g, and 300 ¬µL of clarified lysate was transferred to a new 1.5 mL tube. This was then centrifuged for 10 minutes at 20,000 g. A 100 ¬µL supernatant sample was transferred to 300 ¬µL of Trizol LS, and 400 ¬µL of SPB was added to the pellet as a wash. After another spin at 20,000 g for 10 minutes, the supernatant was removed and the pellet was resuspended by vortexing for 15 minutes in 300 ¬µL of Trizol LS and 100 ¬µL of water. If required, 1 ng of spike-in nanoluc transcript was added to each sample at this step before RNA was isolated using Zymo Direct-Zol RNA extraction columns (Zymo #R2052), and RNA integrity was assessed by the appearance of two sharp rRNA bands on a 1% agarose gel and quantified using the absorbance at 260 nm.
Sequencing libraries were prepared by the University of Chicago Genomics Facility from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215) and Illumina Stranded mRNA Prep (Illumina #20020595) kits.  Paired end 200 bp sequencing was performed on an Illumina NovaSeq 6000.
In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample.
In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.

treatment_protocol: Temperature stresses for sedimentation experiments were completed by centrifuging the culture and exposing the yeast pellet to either 42¬∞C or 46¬∞C water baths for 10 minutes. Control cells were placed inside a 30¬∞C incubator. If cycloheximide treatment was used, cells were pre-treated for 10 minutes with 100 Œºg/mL cycloheximide before heat shock.


title: SedSeq~tg_5~L113~spombe_spikeIn~BY4741~46C~10min~none~BR2~Pellet

status: Public on Apr 26 2024

type: SRA

city: Chicago

first: D Allan

last: Drummond

state: IL

address: 929 E 57th Street

country: USA

institute: University of Chicago

library_source: transcriptomic

molecule: total RNA

organism: Saccharomyces cerevisiae

source_name: yeast cells

growth_protocol: Cells were grown overnight at 30¬∞C with shaking for at least 12 hours to OD600 = 0.4 in synthetic complete dextrose media (SCD) before being exposed to stress.

tag: cell type

value: yeast cells


title: SedSeq~tg_5~L111~spombe_spikeIn~BY4741~46C~10min~none~BR1~Pellet


title: PolySeq~tg_17~L116~PolySeq_0.8%Azide~BY4741~30C~30min~mock~BR2~Free

data_processing:
Sequencing reads were trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename F02 AD-JB-F02_S44_R1_001.fastq.gz AD-JB-F02_S44_R2_001.fastq.gz). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted --readFilesCommand gunzip -c  --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/F02/F02_ --readFilesIn trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz). The estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/F02 --fr-stranded --bootstrap-samples=50 -t 1 trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz).
S. pombe total RNA was added and reads were mapped to saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled.fasta (STAR) using the annotation file saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled_geneid.gff3, and Scerevisiae_orf_coding_all_Scerevisiae_rna_coding_Spombe_cds.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample

extract_protocol:
Around 100 mg of frozen yeast that was collected by vacuum filtration was transferred to a pre-chilled 2 ml Eppendorf Safe-Lock tube. Cells were lysed with a pre-chilled 7 mM stainless steel ball (Retsch #05.368.0035) by 5x90sx30Hz pulses in a Retsch MM100 mixer mill, chilling in liquid nitrogen (LN2) between pulses. Sample was resuspended in 10:1 (v/w) polysome lysis buffer (20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 5 mM MgCl2, 200 Œºg/mL heparin (Sigma #H3149), 1% triton X-100, 0.5 mM TCEP (Goldbio #TCEP25), 100 Œºg/mL cycloheximide (Sigma #C7698-5G), 20 U/ml SUPERase‚Ä¢In (Invitrogen #AM2696), 1:200 Millipore protease inhibitor IV #539136). The lysate was clarified by centrifugation at 3000 g for 30 s, and the clarified lysate was transferred to new tube and aliquots were flash frozen in LN2. A 10‚Äì50% continuous sucrose gradient in polysome gradient buffer (5 mM HEPES-KOH (pH 7.4), 140 mM KCl, 5 mM MgCl2, 100 Œºg/ml cycloheximide, 10 U/ml SUPERase‚Ä¢In, 0.5 mM TCEP) was prepared in SW 28.1 tubes (Seton #7042) using a Biocomp Gradient Master and allowed to cool to 4¬∞C. Clarified lysate (200 ¬µL) was loaded on top of the gradient, and gradients were spun in a SW28.1 rotor at 27,500 rpm for 3.5 hr at 4¬∞C. Gradients were fractionated into 0.6mL fractions using a Biocomp Piston Gradient Fractionator with UV monitoring at 254 nm, and fractions were flash frozen in LN2. UV traces were normalized to the total signal starting with the 40S peak. The samples were generated by pooling 50 ¬µL of each fraction from the free fraction (before the monosome peak) and either separately pooling the fractions with 3+ ribosomes bound and the mono/di-some fractions (for the heat shock experiments), or by combining all ribosome-bound fractions together (azide and ethanol stresses). The spike-in (50 ng of S. pombe total RNA) was then added to each pooled sample. RNA was purified via ethanol precipitation (final concentrations of 0.3 M sodium acetate pH 5.2, 0.3 ¬µg/mL glycoblue (Invitrogen #AM9516), and 70% ethanol) at -20¬∞C overnight followed by centrifugation at 4¬∞C for 30 minutes at 21,000 g. The pellet was washed with 1 mL of 70% ethanol before being resuspended in water. The purified RNA was then treated with Dnase I (NEB) before purifying again using an NEB RNA clean and concentrate kit (NEB #T2030).
Sequencing libraries were prepared by the University of Chicago Genomics Facility from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215) and Illumina Stranded mRNA Prep (Illumina #20020595) kits.  Paired end 200 bp sequencing was performed on an Illumina NovaSeq 6000.
In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample.
In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.


title: 10x3V31_GTM_2956

city: Houston

first: Alice

middle: Yuxuan

last: Tian

email: ayt2@rice.edu, alicet.3993@gmail.com

state: TX

address: One Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Raw sequencing reads were processed using the CellRanger v6.1.2 (10X Genomics) pipeline, against the hg38 reference genome (https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz)
quality control was performed separately for each sample through a quality control pipeline (https://github.com/lijinbio/cellqc). Briefly, empty cells were filtered by dropkick v1.2.8 and ambient RNA was removed using SoupX v1.6.2. Doublets were detected and removed using DoubletFinder. After merging all samples, cells were further filtered manually (features > 300, transcript (UMI) counts > 500, mitochondrial percentage < 5%)
Assembly: hg38
Supplementary files format and content: A .h5ad file of count matrix.

molecule: polyA RNA

organism: Homo sapiens

source_name: primary cell culture

extract_protocol:
Primary hTM cells were isolated from human donor corneal rims deemed unsuitable for transplantation, and characterized. All donor tissues were procured from Saving Sight eye bank (Kansas city, MO). All donor cells used in this study were isolated (between 2012 and 2021), validated, stored in liquid nitrogen, or used in prior studies from our lab. Prior to single cell sequencing, frozen cells were thawed with 15ml pre-warmed media (37C, DMEM:F12=1:1, with 20%FBS, 1%Penn/Step/fungizone) and centrifuged at 300g, 4C for 5min. Cells were washed with media again and suspended in 0.04% BFA and ready for single cell RNA sequencing. Cell viability was checked with trypan blue.
Resuspended single cells were loaded on a 10X Chromium controller for obtaining single cell Gel Beads-In-Emulsions. scRNA-seq libraries were generated using 10X Chromium Single Cell 3’ reagent kits v3.1 (10X Genomics) following the manufacturer’s recommendations (https://www.10xgenomics.com). Sequencing was performed on Illumina Novaseq 6000 (http://www.illumina.com) at the Single Cell Genomics Core at Baylor College of Medicine.

tag: tissue

value: primary cell culture

tag: cell line

value: glaucoma

value: OS

tag: genotype


title: 10x3V31_GTM_1672_P3

description: 10x Genomics

value: unknown


title: SedSeq~tg_5~L110~spombe_spikeIn~BY4741~30C~10min~none~BR1~Pellet


title: 231-09 Plate4 NKB 21

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

data_processing:
The fastq files were aligned to MacaM v7 using STAR aligner v 2.5.2b. The raw counts from ReadsPerGene output of STAR was used for downstream analysis using Seurat v4.0.4.
Assembly: MacaM v7
Supplementary files format and content: The ReadsPerGene output from STAR for each cell was combined as separate columns in ReadCounts.txt.The MacaM v7.8.2 gtf was used to convert gene id to gene names.

organism: Macaca mulatta

source_name: NKB

extract_protocol:
RNA was purified using RNACleanXP Solid Phase Reversible Immobilization (SPRI) beads (Beckman Coulter). Beads with bound RNA were re-suspended in 5 ul of RSB buffer.
Single-cell RNA sequencing was performed by Medgenome using a modified version of the ClonTech SMART-Seq Single Cell Kit that utilized miniaturization to ¼ volumes using a MANTIS Liquid Handler (Formulatrix). Amplified cDNA was fragmented and appended with dual-indexed barcodes using Illumina Nextera kits.

value: NKB


title: SedSeq~tg_5~L112~spombe_spikeIn~BY4741~30C~10min~none~BR2~Sup


title: 231-09 Plate4 NKB 20


title: 231-09 Plate4 NKB 22


title: SedSeq~tg_5~L113~spombe_spikeIn~BY4741~46C~10min~none~BR2~Sup


title: SedSeq~tg_5~L111~spombe_spikeIn~BY4741~46C~10min~none~BR1~Sup


title: SedSeq~tg_5~L113~spombe_spikeIn~BY4741~46C~10min~none~BR2~Total


title: m6A-2

city: Nanjing

first: Shan

last: Gao

address: No. 2 Sipailou, Xuanwu District

country: China

institute: Southeast University

data_processing:
Raw FASTQ files were trimmed by fastp 0.23.2
Reads were mapped to human genome version hg38 by HiSat2 version 2.2.1
MACS2 was used to call peaks
deeptools were used to create bw files
Assembly: hg38
Supplementary files format and content: bigwig, narrow peak
Library strategy: m6A-seq

source_name: clear cell Renal Carcinoma

growth_protocol: 786-O cell lines were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidfied atmosphere with 5%CO2 at 37℃.

extract_protocol:
total cellular RNA extracted from cells were fragmented by RNA fragmentation reagents (Thermo, AM8740). Fragmented RNA was incubated with an anti-m6A antibody (#202003, Synaptic Systems) or IgG IP Grade Rabbit polyclonal antibody at 4°C for 6 h in 500 μl of NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U ml–1 RNase inhibitor).
The purified RNA fragments were used to construct libraries with the SMARTer Stranded Total RNA-seq Kit v2 (Takara)

value: clear cell Renal Carcinoma


title: SedSeq~tg_5~L112~spombe_spikeIn~BY4741~30C~10min~none~BR2~Total


title: m6A-3


title: SedSeq~tg_5~L110~spombe_spikeIn~BY4741~30C~10min~none~BR1~Sup


title: 231-09 Plate4 NKB 1


title: SedSeq~tg_5~L111~spombe_spikeIn~BY4741~46C~10min~none~BR1~Total


title: SedSeq~tg_5~L110~spombe_spikeIn~BY4741~30C~10min~none~BR1~Total


title: 231-09 Plate4 NKB 18


title: SedSeq~tg_9~L109~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR2~Pellet

data_processing:
Sequencing reads were trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename F02 AD-JB-F02_S44_R1_001.fastq.gz AD-JB-F02_S44_R2_001.fastq.gz). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted --readFilesCommand gunzip -c  --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/F02/F02_ --readFilesIn trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz). The estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/F02 --fr-stranded --bootstrap-samples=50 -t 1 trimmed/F02_val_1.fq.gz trimmed/F02_val_2.fq.gz).
NanoLuc spike-in RNA was added and reads were mapped to spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta (STAR) using the annotation file spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3, and spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample

treatment_protocol: Azide stresses were completed at either 0.5% w/v or 0.8% w/v for 30 min in SCD adjusted to pH 6.8 with NaOH. Azide was added from a 10% w/v sodium azide stock in water. Mock treatments were completed by adding pure water at the same volume to cultures.


title: m6A-3 input


title: 231-09 Plate4 NKB 17


title: SedSeq~tg_9~L108~azide3~BY4741~30C~30min~mock~BR2~Pellet


title: m6A-2 input


title: SedSeq~tg_9~L107~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR1~Pellet


title: 231-09 Plate4 NKB 19


title: SedSeq~tg_9~L109~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR2~Sup


title: 231-09 Plate4 NKB 15


title: m6A-1


title: SedSeq~tg_9~L106~azide3~BY4741~30C~30min~mock~BR1~Pellet


title: m6A-1 input


title: SedSeq~tg_9~L108~azide3~BY4741~30C~30min~mock~BR2~Total


title: SedSeq~tg_9~L107~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR1~Total


title: SedSeq~tg_9~L106~azide3~BY4741~30C~30min~mock~BR1~Sup


title: SedSeq~tg_9~L107~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR1~Sup


title: SedSeq~tg_9~L108~azide3~BY4741~30C~30min~mock~BR2~Sup


title: SedSeq~tg_11~L105~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR2~Pellet

treatment_protocol: Ethanol stresses were completed by resuspending centrifuged cell pellets in SCD made with either 5%, 7.5%, 10%, or 15% ethanol for 15 min. Control cells were mock treated by resuspending in normal SCD.


title: SedSeq~tg_11~L104~milderEtOH2~BY4741~30C~15min~mock~BR2~Pellet


title: 231-09 Plate4 NKB 11


title: SedSeq~tg_9~L109~azide3~BY4741~30C~30min~Azide_0.8%_pH6.8~BR2~Total


title: MNG968

city: Seattle

first: Eric

last: Holland

state: WA

address: 1100 Fairview Ave. N

department: Human Biology

institute: Fred Hutch Cancer Center

data_processing:
RNA sequencing reads were aligned to the Gencode GRCh38.primary_assembly genome using STAR2 (v2.7.7a) and then using HTSeq (v0.11.0) reads were counted for each associated gene using the Gencode V39 primary assembly annotations
Gene expression values from combined datasets were batch corrected and normalized and converted to units of log2 transcripts per million (log2TPM)
Assembly: hg38
Supplementary files format and content: tab-delimited files contain log2 TPM values for each sample

source_name: brain

extract_protocol:
RNA was extracted using the Qiagen RNeasy Plus mini kit. Total RNA integrity was checked using an Agilent 4200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA) and quantified using a Trinean DropSense96 spectrophotometer (Caliper Life Sciences, Hopkinton, MA). Samples with RIN < 5 were removed from further analysis. RNA was normalized to 50ng/ul and 500ng was submitted for library preparation.
RNA-seq libraries were prepared from total RNA using the TruSeq Stranded mRNA kit (Illumina, Inc., San Diego, CA, USA). Sequencing was performed using an Illumina NovaSeq 6000 employing a paired-end, 50 base read length (PE50) sequencing strategy. RNA extraction was performed by the Specimen Processing & Research Cell Bank at Fred Hutch. Library preparation and sequencing was performed by the Genomics and Bioinformatics Core Services at Fred Hutch.

value: brain

tag: disease state

value: Meningioma tumor


title: 231-09 Plate4 NKB 12


title: MNG969


title: SedSeq~tg_9~L106~azide3~BY4741~30C~30min~mock~BR1~Total


title: 231-09 Plate4 NKB 16


title: SedSeq~tg_11~L103~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR1~Pellet


title: 231-09 Plate4 NKB 14


title: MNG965


title: MNG967


title: SedSeq~tg_11~L102~milderEtOH2~BY4741~30C~15min~mock~BR1~Pellet


title: MNG966


title: 231-09 Plate4 B 9

source_name: B

value: B


title: MNG964


title: 231-09 Plate4 B 8


title: MNG963


title: SedSeq~tg_11~L102~milderEtOH2~BY4741~30C~15min~mock~BR1~Sup


title: 231-09 Plate4 B 7


title: SedSeq~tg_11~L104~milderEtOH2~BY4741~30C~15min~mock~BR2~Sup


title: SedSeq~tg_11~L105~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR2~Sup


title: MNG962


title: SedSeq~tg_11~L103~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR1~Sup


title: MNG961


title: 231-09 Plate4 NKB 10


title: SedSeq~tg_10~L101~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR2~Pellet


title: 231-09 Plate4 B 4


title: SedSeq~tg_11~L105~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR2~Total


title: SedSeq~tg_10~L100~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR2~Pellet


title: SedSeq~tg_11~L104~milderEtOH2~BY4741~30C~15min~mock~BR2~Total


title: SedSeq~tg_11~L102~milderEtOH2~BY4741~30C~15min~mock~BR1~Total


title: MNG960


title: SedSeq~tg_10~L99~milderEtOH1~BY4741~30C~15min~mock~BR2~Pellet


title: SedSeq~tg_11~L103~milderEtOH2~BY4741~30C~15min~EtOH_7.5%~BR1~Total


title: 231-09 Plate4 B 22


title: 231-09 Plate4 B 2


title: MNG959


title: SedSeq~tg_10~L97~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR1~Pellet


title: 231-09 Plate4 B 21


title: SedSeq~tg_10~L101~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR2~Sup


title: 231-09 Plate4 B 5


title: MNG957


title: SedSeq~tg_10~L98~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR1~Pellet


title: MNG955


title: SedSeq~tg_10~L98~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR1~Sup


title: MNG956


title: 231-09 Plate4 B 20


title: 231-09 Plate4 B 1


title: MNG954


title: SedSeq~tg_10~L100~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR2~Total


title: SedSeq~tg_10~L96~milderEtOH1~BY4741~30C~15min~mock~BR1~Pellet


title: SedSeq~tg_10~L99~milderEtOH1~BY4741~30C~15min~mock~BR2~Total


title: 231-09 Plate4 B 16


title: MNG952


title: MNG958


title: SedSeq~tg_10~L98~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR1~Total


title: 231-09 Plate4 B 18


title: SedSeq~tg_10~L96~milderEtOH1~BY4741~30C~15min~mock~BR1~Total


title: SedSeq~tg_10~L96~milderEtOH1~BY4741~30C~15min~mock~BR1~Sup


title: 231-09 Plate4 B 19


title: SedSeq~tg_2~L95~hairpinReporters~yHG027~30C~NA~none~BR1~Pellet


title: SedSeq~tg_10~L97~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR1~Total


title: SedSeq~tg_10~L100~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR2~Sup


title: 231-09 Plate4 B 13


title: 231-09 Plate4 B 12


title: SedSeq~tg_2~L93~hairpinReporters~yHG008~30C~NA~none~BR1~Pellet


title: SedSeq~tg_10~L97~milderEtOH1~BY4741~30C~15min~EtOH_5%~BR1~Sup


title: SedSeq~tg_10~L99~milderEtOH1~BY4741~30C~15min~mock~BR2~Sup


title: SedSeq~tg_2~L94~hairpinReporters~yHG026~30C~NA~none~BR1~Pellet


title: MNG951


title: SedSeq~tg_10~L101~milderEtOH1~BY4741~30C~15min~EtOH_10%~BR2~Total


title: 231-09 Plate4 B 11


title: MNG953


title: SedSeq~tg_2~L92~hairpinReporters~yHG007~30C~NA~none~BR1~Pellet


title: MNG949


title: SedSeq~tg_2~L91~hairpinReporters~yHG006~30C~NA~none~BR1~Pellet


title: 231-09 Plate4 B 10


title: 237-09 Plate2 NK 9

source_name: NK

value: NK


title: SedSeq~tg_2~L94~hairpinReporters~yHG026~30C~NA~none~BR1~Sup


title: SedSeq~tg_2~L89~hairpinReporters~yHG010~30C~NA~none~BR1~Pellet


title: MNG948


title: MNG950


title: 237-09 Plate2 NK 8


title: SedSeq~tg_2~L95~hairpinReporters~yHG027~30C~NA~none~BR1~Sup


title: SedSeq~tg_2~L93~hairpinReporters~yHG008~30C~NA~none~BR1~Sup


title: MNG947


title: 237-09 Plate2 NK 7


title: SedSeq~tg_2~L91~hairpinReporters~yHG006~30C~NA~none~BR1~Sup


title: 237-09 Plate2 NK 6


title: MNG945


title: SedSeq~tg_2~L90~hairpinReporters~yHG005~30C~NA~none~BR1~Sup


title: SedSeq~tg_2~L89~hairpinReporters~yHG010~30C~NA~none~BR1~Sup


title: SedSeq~tg_2~L95~hairpinReporters~yHG027~30C~NA~none~BR1~Total


title: SedSeq~tg_2~L93~hairpinReporters~yHG008~30C~NA~none~BR1~Total


title: MNG944


title: SedSeq~tg_2~L94~hairpinReporters~yHG026~30C~NA~none~BR1~Total


title: SedSeq~tg_2~L90~hairpinReporters~yHG005~30C~NA~none~BR1~Pellet


title: MNG943


title: 237-09 Plate2 NK 4


title: SedSeq~tg_2~L91~hairpinReporters~yHG006~30C~NA~none~BR1~Total


title: SedSeq~tg_2~L92~hairpinReporters~yHG007~30C~NA~none~BR1~Total


title: SedSeq~tg_2~L90~hairpinReporters~yHG005~30C~NA~none~BR1~Total


title: MNG942


title: 237-09 Plate2 NK 3


title: SedSeq~tg_2~L92~hairpinReporters~yHG007~30C~NA~none~BR1~Sup


title: MNG946


title: SedSeq~tg_2~L89~hairpinReporters~yHG010~30C~NA~none~BR1~Total


title: MNG941


title: SedSeq~tg_20~L84~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR2~Pellet

data_processing:
The unique molecular indices (UMIs) were extracted from fastq R2 using Umi-Tools v1.1.4(Smith et al. 2017) and stored in fastq R1 and R3 (e.g. umi_tools extract --bc-pattern=XXXXXXXXNNNNNNNNNNN -I AD-JB-1S-HG02_S2_R2_001.fastq.gz --read2-in=AD-JB-1S-HG02_S2_R1_001.fastq.gz --read2-out=labeled_fastq/HG002/HG002_R1.umi.fastq. Sequencing reads were then trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --gzip --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename HG002 labeled_fastq/HG002/HG002_R1.umi.fastq labeled_fastq/HG002/HG002_R3.umi.fastq). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted  --readFilesCommand gunzip -c --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/HG002/HG002_ --readFilesIn trimmed/HG002_val_1.fq.gz trimmed/HG002_val_2.fq.gz). Umi-Tools was then used again to deduplicate the reads (e.g. umi_tools dedup --stdin=mapped_reads/HG002/HG002_Aligned_Sorted.out.bam  --chimeric-pairs=discard --unpaired-reads=discard --spliced-is-unique --paired -S mapped_reads/HG002/HG002_Aligned.sortedByCoord.dedup.out.bam). The reads were split again into fastq files using samtools v1.16.1(Li et al. 2009), and then estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/HG002 --rf-stranded --bootstrap-samples=50 -t 1 mapped_reads/HG002/HG002_Aligned_dedup_R1.fastq.gz mapped_reads/HG002/HG002_Aligned_dedup_R3.fastq.gz).
NanoLuc spike-in RNA was added and reads were mapped to spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta (STAR) using the annotation file spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3, and spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample

extract_protocol:
Biochemical fractionation was completed similarly to Wallace et al. [2015], with the major exception that 20,000 g for 10 min was used rather than the original 100,000 g for 20 min. In short, 50 mL cultures of treated yeast were harvested by centrifugation at 3000 g for 5 minutes, then resuspended in 100 ¬µL of soluble protein buffer (SPB: 20 mM HEPES, pH 7.4, 140 mM KCl, 2 mM EDTA, 0.1 mM TCEP, 1:200 protease inhibitor (Millipore #539136), 1:1000 SUPERase‚Ä¢In RNase Inhibitor (Invitrogen #AM2696)), and flash frozen in liquid nitrogen as a pellet in a 2 mL Eppendorf Safe-Lock tube (Eppendorf #0030123620) with a 7 mm steel ball (Retsch #05.368.0035). The cells were then lysed using a Retsch MM400 for 5x90s at 30 Hz, chilling in liquid nitrogen between each shaking repeat. The lysed cells were resuspended in 600 ¬µL of SPB, and 100 ¬µL of total sample was transferred to 300 ¬µL of Trizol LS (Invitrogen #10296010). The remainder was centrifuged for 30 seconds at 3000 g, and 300 ¬µL of clarified lysate was transferred to a new 1.5 mL tube. This was then centrifuged for 10 minutes at 20,000 g. A 100 ¬µL supernatant sample was transferred to 300 ¬µL of Trizol LS, and 400 ¬µL of SPB was added to the pellet as a wash. After another spin at 20,000 g for 10 minutes, the supernatant was removed and the pellet was resuspended by vortexing for 15 minutes in 300 ¬µL of Trizol LS and 100 ¬µL of water. If required, 1 ng of spike-in nanoluc transcript was added to each sample at this step before RNA was isolated using Zymo Direct-Zol RNA extraction columns (Zymo #R2052), and RNA integrity was assessed by the appearance of two sharp rRNA bands on a 1% agarose gel and quantified using the absorbance at 260 nm.
Sequencing libraries were prepared by from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215), NEBNext Multiplex Oligos (UMI Adaptor RNA Set 1, NEB #E7335L) and NEBnext Ultra II Directional RNA library prep kits (NEB #E7760L).  Paired end 200 bp sequencing with additional reads for dual 8/8 indices plus the 11nt UMI after the i7 index was performed on an Illumina NovaSeq 6000 at the University of Chicago Genomics Facility.
In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample.
In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.


title: MNG940


title: SedSeq~tg_20~L82~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR1~Pellet


title: SedSeq~tg_20~L83~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR2~Pellet


title: SedSeq~tg_20~L81~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR1~Pellet


title: 237-09 Plate2 NK 2


title: 237-09 Plate2 NK 22


title: 237-09 Plate2 NK 5


title: SedSeq~tg_3~L80~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR2~Pellet

treatment_protocol: For depletion experiments, yeast were grown at 30¬∞C in YPD to OD600 = 0.1. To induce TIR1 ligase, 5 ¬µM Œ≤-estradiol (10 mM stock in DMSO) or an equivalent volume of DMSO (for mock treatment) was added to each culture and they were incubated for 75 minutes. To induce degradation, either 100 ¬µM of Indole-3-acetic acid sodium salt (Sigma #I5148, 250 mM stock in DMSO) or 5 ¬µM of 5-Ph-IAA (Medchemexpress #HY-134653, 5 mM stock in DMSO) was added. After 2 hours of auxin exposure, the culture was centrifuged and the yeast pellet was incubated in either 42¬∞C or 46¬∞C water baths for 10 minutes. Control cells were placed inside a 30¬∞C incubator.


title: 237-09 Plate2 NK 21


title: SedSeq~tg_3~L79~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR2~Pellet


title: MNG939


title: MNG937


title: MNG938


title: SedSeq~tg_3~L78~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR2~Pellet


title: 237-09 Plate2 NK 20


title: SedSeq~tg_3~L77~eIF3b_deplete~yJB042~46C~10min~mock~BR2~Pellet


title: SedSeq~tg_3~L74~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR1~Pellet


title: SedSeq~tg_3~L76~eIF3b_deplete~yJB042~42C~10min~mock~BR2~Pellet


title: 237-09 Plate2 NK 1


title: MNG935


title: SedSeq~tg_3~L75~eIF3b_deplete~yJB042~30C~10min~mock~BR2~Pellet


title: SedSeq~tg_3~L72~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR1~Pellet


title: SedSeq~tg_3~L71~eIF3b_deplete~yJB042~46C~10min~mock~BR1~Pellet


title: 237-09 Plate2 NK 18


title: SedSeq~tg_3~L70~eIF3b_deplete~yJB042~42C~10min~mock~BR1~Pellet


title: SedSeq~tg_3~L73~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR1~Pellet


title: 237-09 Plate2 NK 19


title: 237-09 Plate2 NK 17


title: MNG934


title: SedSeq~tg_3~L69~eIF3b_deplete~yJB042~30C~10min~mock~BR1~Pellet


title: 237-09 Plate2 NK 16


title: SedSeq~tg_4~L68~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR2~Pellet


title: SedSeq~tg_4~L66~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR2~Pellet


title: 237-09 Plate2 NK 15


title: SedSeq~tg_4~L67~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR2~Pellet


title: MNG931


title: SedSeq~tg_4~L64~eIF4E_deplete~yHG038~42C~10min~mock~BR2~Pellet


title: 237-09 Plate2 NK 14


title: MNG936


title: MNG932


title: 237-09 Plate2 NK 13


title: SedSeq~tg_4~L62~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR1~Pellet


title: SedSeq~tg_4~L65~eIF4E_deplete~yHG038~46C~10min~mock~BR2~Pellet


title: MNG930


title: MNG929


title: MNG928


title: 237-09 Plate2 NK 12


title: MNG927


title: 237-09 Plate2 NK 10


title: MNG926


title: MNG925


title: SedSeq~tg_4~L63~eIF4E_deplete~yHG038~30C~10min~mock~BR2~Pellet


title: 237-09 Plate2 NKB 9


title: MNG933


title: 237-09 Plate2 NKB 8


title: 237-09 Plate2 NK 11


title: MNG924


title: 237-09 Plate2 NKB 7


title: MNG923


title: 237-09 Plate2 NKB 6


title: SedSeq~tg_20~L83~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR2~Sup


title: SedSeq~tg_3~L79~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR2~Sup


title: SedSeq~tg_20~L84~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR2~Sup


title: SedSeq~tg_20~L81~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR1~Sup


title: SedSeq~tg_3~L75~eIF3b_deplete~yJB042~30C~10min~mock~BR2~Sup


title: SedSeq~tg_3~L76~eIF3b_deplete~yJB042~42C~10min~mock~BR2~Sup


title: SedSeq~tg_3~L78~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR2~Sup


title: SedSeq~tg_4~L59~eIF4E_deplete~yHG038~46C~10min~mock~BR1~Pellet


title: SedSeq~tg_3~L80~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR2~Sup


title: MNG922


title: SedSeq~tg_3~L77~eIF3b_deplete~yJB042~46C~10min~mock~BR2~Sup


title: SedSeq~tg_20~L82~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR1~Sup


title: SedSeq~tg_4~L58~eIF4E_deplete~yHG038~42C~10min~mock~BR1~Pellet


title: SedSeq~tg_4~L60~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR1~Pellet


title: SedSeq~tg_3~L74~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR1~Sup


title: SedSeq~tg_4~L57~eIF4E_deplete~yHG038~30C~10min~mock~BR1~Pellet


title: SedSeq~tg_3~L73~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR1~Sup


title: MNG921


title: 237-09 Plate2 NKB 5


title: SedSeq~tg_4~L61~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR1~Pellet


title: MNG920


title: SedSeq~tg_3~L72~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR1~Sup


title: SedSeq~tg_3~L71~eIF3b_deplete~yJB042~46C~10min~mock~BR1~Sup


title: 237-09 Plate2 NKB 4


title: SedSeq~tg_3~L70~eIF3b_deplete~yJB042~42C~10min~mock~BR1~Sup


title: 237-09 Plate2 NKB 44


title: MNG919


title: SedSeq~tg_3~L69~eIF3b_deplete~yJB042~30C~10min~mock~BR1~Sup


title: 237-09 Plate2 NKB 43


title: SedSeq~tg_4~L65~eIF4E_deplete~yHG038~46C~10min~mock~BR2~Sup


title: SedSeq~tg_4~L62~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR1~Sup


title: 237-09 Plate2 NKB 40


title: MNG915


title: SedSeq~tg_4~L60~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR1~Sup


title: MNG916


title: SedSeq~tg_4~L64~eIF4E_deplete~yHG038~42C~10min~mock~BR2~Sup


title: 237-09 Plate2 NKB 42


title: SedSeq~tg_4~L66~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR2~Sup


title: MNG913


title: SedSeq~tg_4~L61~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR1~Sup


title: SedSeq~tg_4~L63~eIF4E_deplete~yHG038~30C~10min~mock~BR2~Sup


title: MNG914


title: 237-09 Plate2 NKB 41


title: SedSeq~tg_20~L84~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR2~Total


title: MNG917


title: SedSeq~tg_4~L58~eIF4E_deplete~yHG038~42C~10min~mock~BR1~Sup


title: SedSeq~tg_4~L57~eIF4E_deplete~yHG038~30C~10min~mock~BR1~Sup


title: 237-09 Plate2 NKB 3


title: 237-09 Plate2 NKB 38


title: 237-09 Plate2 NKB 37


title: SedSeq~tg_20~L83~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR2~Total


title: SedSeq~tg_20~L82~Azide_0.5%_pH6.8~BY4741~30C~30min~Azide_0.5%_pH6.8~BR1~Total


title: MNG912


title: SedSeq~tg_4~L68~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR2~Sup


title: MNG918


title: SedSeq~tg_4~L67~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR2~Sup


title: 237-09 Plate2 NKB 39


title: SedSeq~tg_20~L81~Azide_0.5%_pH6.8~BY4741~30C~30min~mock~BR1~Total


title: SedSeq~tg_4~L59~eIF4E_deplete~yHG038~46C~10min~mock~BR1~Sup


title: MNG911


title: SedSeq~tg_3~L80~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR2~Total


title: SedSeq~tg_3~L79~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR2~Total


title: SedSeq~tg_3~L77~eIF3b_deplete~yJB042~46C~10min~mock~BR2~Total


title: 237-09 Plate2 NKB 36


title: MNG909


title: 237-09 Plate2 NKB 34


title: MNG910


title: SedSeq~tg_3~L75~eIF3b_deplete~yJB042~30C~10min~mock~BR2~Total


title: MNG907


title: 237-09 Plate2 NKB 33


title: 237-09 Plate2 NKB 35


title: 237-09 Plate2 NKB 32


title: SedSeq~tg_3~L76~eIF3b_deplete~yJB042~42C~10min~mock~BR2~Total


title: SedSeq~tg_3~L71~eIF3b_deplete~yJB042~46C~10min~mock~BR1~Total


title: SedSeq~tg_3~L74~eIF3b_deplete~yJB042~46C~10min~eIF3b_deplete~BR1~Total


title: SedSeq~tg_3~L72~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR1~Total


title: MNG906


title: SedSeq~tg_3~L70~eIF3b_deplete~yJB042~42C~10min~mock~BR1~Total


title: MNG905


title: SedSeq~tg_3~L73~eIF3b_deplete~yJB042~42C~10min~eIF3b_deplete~BR1~Total


title: SedSeq~tg_3~L69~eIF3b_deplete~yJB042~30C~10min~mock~BR1~Total


title: SedSeq~tg_4~L68~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR2~Total


title: 237-09 Plate2 NKB 30


title: MNG908


title: MNG904


title: SedSeq~tg_3~L78~eIF3b_deplete~yJB042~30C~10min~eIF3b_deplete~BR2~Total


title: SedSeq~tg_4~L67~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR2~Total


title: SedSeq~tg_4~L66~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR2~Total


title: 237-09 Plate2 NKB 2


title: SedSeq~tg_4~L65~eIF4E_deplete~yHG038~46C~10min~mock~BR2~Total


title: SedSeq~tg_4~L64~eIF4E_deplete~yHG038~42C~10min~mock~BR2~Total


title: MNG903


title: MNG901


title: MNG902


title: SedSeq~tg_4~L60~eIF4E_deplete~yHG038~30C~10min~eIF4E_deplete~BR1~Total


title: 237-09 Plate2 NKB 28


title: 237-09 Plate2 NKB 29


title: 237-09 Plate2 NKB 31


title: SedSeq~tg_4~L62~eIF4E_deplete~yHG038~46C~10min~eIF4E_deplete~BR1~Total


title: MNG900


title: SedSeq~tg_4~L58~eIF4E_deplete~yHG038~42C~10min~mock~BR1~Total


title: SedSeq~tg_4~L63~eIF4E_deplete~yHG038~30C~10min~mock~BR2~Total


title: SedSeq~tg_4~L57~eIF4E_deplete~yHG038~30C~10min~mock~BR1~Total


title: 237-09 Plate2 NKB 25


title: SedSeq~tg_4~L59~eIF4E_deplete~yHG038~46C~10min~mock~BR1~Total


title: PolySeq~tg_15~L56~PolySeq_10min~BY4741~46C~10min~none~BR2~Poly

data_processing:
The unique molecular indices (UMIs) were extracted from fastq R2 using Umi-Tools v1.1.4(Smith et al. 2017) and stored in fastq R1 and R3 (e.g. umi_tools extract --bc-pattern=XXXXXXXXNNNNNNNNNNN -I AD-JB-1S-HG02_S2_R2_001.fastq.gz --read2-in=AD-JB-1S-HG02_S2_R1_001.fastq.gz --read2-out=labeled_fastq/HG002/HG002_R1.umi.fastq. Sequencing reads were then trimmed using TrimGalore (v0.6.10, https://github.com/FelixKrueger/TrimGalore) using default settings (e.g. trim_galore --paired --gzip --fastqc_args '--outdir fastqc/' -j 4 -o trimmed --basename HG002 labeled_fastq/HG002/HG002_R1.umi.fastq labeled_fastq/HG002/HG002_R3.umi.fastq). They were mapped using STAR v2.7.10b(Dobin et al. 2013) (e.g. STAR --outSAMtype BAM Unsorted  --readFilesCommand gunzip -c --sjdbGTFfile spike_saccharomyces_cerevisiae_R64-3-1_20210421_geneid.gff3 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon CDS --sjdbGTFtagExonParentGene gene_id --runThreadN 4 --alignMatesGapMax 20000 --limitBAMsortRAM 1445804817 --genomeDir STAR_spike_saccharomyces_cerevisiae_R64-3-1_20210421 --outFileNamePrefix mapped_reads/HG002/HG002_ --readFilesIn trimmed/HG002_val_1.fq.gz trimmed/HG002_val_2.fq.gz). Umi-Tools was then used again to deduplicate the reads (e.g. umi_tools dedup --stdin=mapped_reads/HG002/HG002_Aligned_Sorted.out.bam  --chimeric-pairs=discard --unpaired-reads=discard --spliced-is-unique --paired -S mapped_reads/HG002/HG002_Aligned.sortedByCoord.dedup.out.bam). The reads were split again into fastq files using samtools v1.16.1(Li et al. 2009), and then estimated counts and TPMs were generated using kallisto v0.48.0(Bray et al. 2016) (e.g. kallisto quant -i spike_Scerevisiae_orf_coding_all_Scerevisiae_rna_coding.fasta.idx -o kallisto_quant/HG002 --rf-stranded --bootstrap-samples=50 -t 1 mapped_reads/HG002/HG002_Aligned_dedup_R1.fastq.gz mapped_reads/HG002/HG002_Aligned_dedup_R3.fastq.gz).
S. pombe total RNA was added and reads were mapped to saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled.fasta (STAR) using the annotation file saccharomyces_cerevisiae_R64-3-1_20210421_Schizosaccharomyces_pombe_all_chromosomes_relabelled_geneid.gff3, and Scerevisiae_orf_coding_all_Scerevisiae_rna_coding_Spombe_cds.fasta (kallisto).
In general, sequencing reads were mapped to the transcriptome using kallisto to extract estimated counts and TPMs.
Assembly: S288C R64.3.1
Supplementary files format and content: estimated counts and TPMs per RNA sample

extract_protocol:
Around 100 mg of frozen yeast that was collected by vacuum filtration was transferred to a pre-chilled 2 ml Eppendorf Safe-Lock tube. Cells were lysed with a pre-chilled 7 mM stainless steel ball (Retsch #05.368.0035) by 5x90sx30Hz pulses in a Retsch MM100 mixer mill, chilling in liquid nitrogen (LN2) between pulses. Sample was resuspended in 10:1 (v/w) polysome lysis buffer (20 mM HEPES-KOH (pH 7.4), 100 mM KCl, 5 mM MgCl2, 200 Œºg/mL heparin (Sigma #H3149), 1% triton X-100, 0.5 mM TCEP (Goldbio #TCEP25), 100 Œºg/mL cycloheximide (Sigma #C7698-5G), 20 U/ml SUPERase‚Ä¢In (Invitrogen #AM2696), 1:200 Millipore protease inhibitor IV #539136). The lysate was clarified by centrifugation at 3000 g for 30 s, and the clarified lysate was transferred to new tube and aliquots were flash frozen in LN2. A 10‚Äì50% continuous sucrose gradient in polysome gradient buffer (5 mM HEPES-KOH (pH 7.4), 140 mM KCl, 5 mM MgCl2, 100 Œºg/ml cycloheximide, 10 U/ml SUPERase‚Ä¢In, 0.5 mM TCEP) was prepared in SW 28.1 tubes (Seton #7042) using a Biocomp Gradient Master and allowed to cool to 4¬∞C. Clarified lysate (200 ¬µL) was loaded on top of the gradient, and gradients were spun in a SW28.1 rotor at 27,500 rpm for 3.5 hr at 4¬∞C. Gradients were fractionated into 0.6mL fractions using a Biocomp Piston Gradient Fractionator with UV monitoring at 254 nm, and fractions were flash frozen in LN2. UV traces were normalized to the total signal starting with the 40S peak. The samples were generated by pooling 50 ¬µL of each fraction from the free fraction (before the monosome peak) and either separately pooling the fractions with 3+ ribosomes bound and the mono/di-some fractions (for the heat shock experiments), or by combining all ribosome-bound fractions together (azide and ethanol stresses). The spike-in (50 ng of S. pombe total RNA) was then added to each pooled sample. RNA was purified via ethanol precipitation (final concentrations of 0.3 M sodium acetate pH 5.2, 0.3 ¬µg/mL glycoblue (Invitrogen #AM9516), and 70% ethanol) at -20¬∞C overnight followed by centrifugation at 4¬∞C for 30 minutes at 21,000 g. The pellet was washed with 1 mL of 70% ethanol before being resuspended in water. The purified RNA was then treated with Dnase I (NEB) before purifying again using an NEB RNA clean and concentrate kit (NEB #T2030).
Sequencing libraries were prepared by from DNase I treated RNA using Qiagen FastSelect (Qiagen #334215), NEBNext Multiplex Oligos (UMI Adaptor RNA Set 1, NEB #E7335L) and NEBnext Ultra II Directional RNA library prep kits (NEB #E7760L).  Paired end 200 bp sequencing with additional reads for dual 8/8 indices plus the 11nt UMI after the i7 index was performed on an Illumina NovaSeq 6000 at the University of Chicago Genomics Facility.
In general, cells were first lysed by cryo-grinding with a steel ball. When appropriate, fractionation by sedimentation or sucrose gradients was performed and RNA was dissolved in Trizol. The RNA was then purified using Zymo Direct-Zol columns. For more details, please see the protocol associated with each sample.
In general, RNA sequencing libraries were prepared using either Illumina RNA sequencing kits or NEBNext Ultra II RNA sequencing kits.

treatment_protocol: Temperature stresses for Polysome-seq experiments were completed by collecting yeast via vacuum filtration onto a 0.45 Œºm filter, putting the filter in 125 mL of pre-warmed media and incubating in a temperature controlled shaking water bath or incubator. After the indicated time, samples were harvested again via vacuum filtration and immediately scraped into liquid nitrogen.


title: MNG899


title: MNG898


title: 237-09 Plate2 NKB 27


title: PolySeq~tg_15~L55~PolySeq_10min~BY4741~42C~10min~none~BR2~Poly


title: PolySeq~tg_15~L53~PolySeq_10min~BY4741~46C~10min~none~BR1~Poly


title: 237-09 Plate2 NKB 24


title: PolySeq~tg_15~L54~PolySeq_10min~BY4741~30C~10min~none~BR2~Poly


title: PolySeq~tg_15~L52~PolySeq_10min~BY4741~42C~10min~none~BR1~Poly


title: MNG897


title: SedSeq~tg_4~L61~eIF4E_deplete~yHG038~42C~10min~eIF4E_deplete~BR1~Total


title: 237-09 Plate2 NKB 26


title: 237-09 Plate2 NKB 23


title: PolySeq~tg_15~L51~PolySeq_10min~BY4741~30C~10min~none~BR1~Poly


title: PolySeq~tg_15~L56~PolySeq_10min~BY4741~46C~10min~none~BR2~Mono


title: 237-09 Plate2 NKB 22


title: MNG895


title: MNG896


title: PolySeq~tg_15~L55~PolySeq_10min~BY4741~42C~10min~none~BR2~Mono


title: 237-09 Plate2 NKB 21


title: PolySeq~tg_15~L53~PolySeq_10min~BY4741~46C~10min~none~BR1~Mono


title: MNG894


title: 237-09 Plate2 NKB 20


title: MNG893


title: PolySeq~tg_15~L51~PolySeq_10min~BY4741~30C~10min~none~BR1~Mono


title: PolySeq~tg_15~L54~PolySeq_10min~BY4741~30C~10min~none~BR2~Mono


title: 237-09 Plate2 NKB 1


title: PolySeq~tg_15~L56~PolySeq_10min~BY4741~46C~10min~none~BR2~Free


title: PolySeq~tg_15~L55~PolySeq_10min~BY4741~42C~10min~none~BR2~Free


title: MNG892


title: PolySeq~tg_15~L54~PolySeq_10min~BY4741~30C~10min~none~BR2~Free


title: PolySeq~tg_15~L53~PolySeq_10min~BY4741~46C~10min~none~BR1~Free


title: 237-09 Plate2 NKB 19


title: MNG370


title: PolySeq~tg_15~L51~PolySeq_10min~BY4741~30C~10min~none~BR1~Free


title: 237-09 Plate2 NKB 18


title: MNG369


title: PolySeq~tg_15~L56~PolySeq_10min~BY4741~46C~10min~none~BR2~Total


title: PolySeq~tg_15~L55~PolySeq_10min~BY4741~42C~10min~none~BR2~Total


title: 237-09 Plate2 NKB 17


title: MNG368


title: PolySeq~tg_15~L52~PolySeq_10min~BY4741~42C~10min~none~BR1~Mono


title: 237-09 Plate2 NKB 16


title: PolySeq~tg_15~L54~PolySeq_10min~BY4741~30C~10min~none~BR2~Total


title: PolySeq~tg_15~L53~PolySeq_10min~BY4741~46C~10min~none~BR1~Total


title: MNG367


title: 237-09 Plate2 NKB 15


title: MNG366


title: 237-09 Plate2 NKB 14


title: MNG365


title: PolySeq~tg_15~L51~PolySeq_10min~BY4741~30C~10min~none~BR1~Total


title: SedSeq~tg_13~L50~uprInduction~BY4741~30C~15min~DTT_10mM~BR2~Pellet

treatment_protocol: DTT stress was completed by adding 10 mM DTT for 15 minutes prior to harvesting


title: PolySeq~tg_15~L52~PolySeq_10min~BY4741~42C~10min~none~BR1~Free


title: SedSeq~tg_13~L48~uprInduction~BY4741~30C~15min~mock~BR2~Pellet


title: SedSeq~tg_13~L47~uprInduction~BY4741~30C~15min~mock~BR1~Pellet


title: 237-09 Plate2 NKB 12


title: 237-09 Plate2 NKB 13


title: MNG363


title: SedSeq~tg_13~L47~uprInduction~BY4741~30C~15min~mock~BR1~Sup


title: SedSeq~tg_13~L50~uprInduction~BY4741~30C~15min~DTT_10mM~BR2~Sup


title: MNG362


title: SedSeq~tg_13~L50~uprInduction~BY4741~30C~15min~DTT_10mM~BR2~Total


title: SedSeq~tg_13~L48~uprInduction~BY4741~30C~15min~mock~BR2~Sup


title: 237-09 Plate2 NKB 10


title: 237-09 Plate2 NKB 11


title: MNG361


title: SedSeq~tg_13~L49~uprInduction~BY4741~30C~15min~DTT_10mM~BR1~Total


title: SedSeq~tg_13~L48~uprInduction~BY4741~30C~15min~mock~BR2~Total


title: SedSeq~tg_13~L49~uprInduction~BY4741~30C~15min~DTT_10mM~BR1~Pellet


title: 237-09 Plate2 B 8


title: PolySeq~tg_15~L52~PolySeq_10min~BY4741~42C~10min~none~BR1~Total


title: SedSeq~tg_13~L47~uprInduction~BY4741~30C~15min~mock~BR1~Total


title: MNG360


title: SedSeq~tg_12~L46~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR2~Pellet


title: SedSeq~tg_12~L44~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR2~Pellet


title: MNG364


title: MNG359


title: SedSeq~tg_12~L43~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR1~Pellet


title: SedSeq~tg_12~L41~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR1~Pellet


title: SedSeq~tg_12~L46~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR2~Sup


title: 237-09 Plate2 B 6


title: SedSeq~tg_13~L49~uprInduction~BY4741~30C~15min~DTT_10mM~BR1~Sup


title: MNG358


title: SedSeq~tg_12~L43~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR1~Sup


title: MNG356


title: 237-09 Plate2 B 3


title: SedSeq~tg_12~L41~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR1~Sup


title: SedSeq~tg_12~L43~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR1~Total


title: SedSeq~tg_12~L44~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR2~Total


title: 237-09 Plate2 B 4


title: MNG355


title: 237-09 Plate2 B 7


title: SedSeq~tg_12~L41~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR1~Total


title: SedSeq~tg_12~L46~HyperOsmotic_EtOH~BY4741~30C~15min~EtOH_15%~BR2~Total


title: SedSeq~tg_19~L40~CHX~BY4741~46C~10min~CHX~BR2~Pellet


title: MNG354


title: MNG353


title: 237-09 Plate2 B 22


title: 237-09 Plate2 B 2


title: SedSeq~tg_19~L38~CHX~BY4741~30C~10min~CHX~BR2~Pellet


title: SedSeq~tg_12~L44~HyperOsmotic_EtOH~BY4741~30C~15min~mock~BR2~Sup


title: MNG357


title: MNG352


title: 237-09 Plate2 B 5


title: SedSeq~tg_19~L36~CHX~BY4741~42C~10min~mock~BR2~Pellet


title: SedSeq~tg_19~L37~CHX~BY4741~46C~10min~mock~BR2~Pellet


title: 237-09 Plate2 B 20


title: 237-09 Plate2 B 1


title: SedSeq~tg_19~L35~CHX~BY4741~30C~10min~mock~BR2~Pellet


title: SedSeq~tg_19~L40~CHX~BY4741~46C~10min~CHX~BR2~Sup


title: 237-09 Plate2 B 19


title: SedSeq~tg_19~L38~CHX~BY4741~30C~10min~CHX~BR2~Sup


title: SedSeq~tg_19~L37~CHX~BY4741~46C~10min~mock~BR2~Sup


title: MNG350


title: SedSeq~tg_19~L39~CHX~BY4741~42C~10min~CHX~BR2~Sup


title: 237-09 Plate2 B 18


title: MNG349


title: SedSeq~tg_19~L39~CHX~BY4741~42C~10min~CHX~BR2~Pellet


title: 237-09 Plate2 B 21


title: MNG348


title: SedSeq~tg_19~L40~CHX~BY4741~46C~10min~CHX~BR2~Total


title: SedSeq~tg_19~L36~CHX~BY4741~42C~10min~mock~BR2~Sup


title: SedSeq~tg_19~L35~CHX~BY4741~30C~10min~mock~BR2~Sup


title: 237-09 Plate2 B 17


title: SedSeq~tg_19~L39~CHX~BY4741~42C~10min~CHX~BR2~Total


title: 237-09 Plate2 B 16


title: MNG347


title: SedSeq~tg_19~L38~CHX~BY4741~30C~10min~CHX~BR2~Total


title: MNG351


title: SedSeq~tg_19~L37~CHX~BY4741~46C~10min~mock~BR2~Total


title: SedSeq~tg_19~L36~CHX~BY4741~42C~10min~mock~BR2~Total


title: MNG346


title: 237-09 Plate2 B 14


title: SedSeq~tg_19~L33~CHX~BY4741~30C~10min~CHX~BR1~Pellet


title: 237-09 Plate2 B 15


title: SedSeq~tg_19~L35~CHX~BY4741~30C~10min~mock~BR2~Total


title: SedSeq~tg_19~L34~CHX~BY4741~46C~10min~CHX~BR1~Pellet


title: 237-09 Plate2 B 13


title: SedSeq~tg_19~L32~CHX~BY4741~46C~10min~mock~BR1~Pellet


title: SedSeq~tg_19~L34~CHX~BY4741~46C~10min~CHX~BR1~Sup


title: 237-09 Plate2 B 12


title: MNG343


title: MNG342


title: SedSeq~tg_19~L33~CHX~BY4741~30C~10min~CHX~BR1~Sup


title: SedSeq~tg_19~L32~CHX~BY4741~46C~10min~mock~BR1~Sup


title: SedSeq~tg_19~L31~CHX~BY4741~30C~10min~mock~BR1~Sup


title: MNG341


title: 237-09 Plate2 B 10


title: 237-09 Plate2 B 11


title: SedSeq~tg_19~L32~CHX~BY4741~46C~10min~mock~BR1~Total


title: MNG340


title: MNG345


title: SedSeq~tg_19~L34~CHX~BY4741~46C~10min~CHX~BR1~Total


title: MNG339


title: SedSeq~tg_19~L31~CHX~BY4741~30C~10min~mock~BR1~Total


title: MNG344


title: 237-09 Plate1 NK 7


title: MNG338


title: Small screen on Day 25 Dox+

city: New York

first: Hyein

middle: Sophia

last: Cho

state: NY

address: 430 E 67th St

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

description: 220929 small pool gRNA raw and processed figures.xlsx

library_source: genomic

data_processing:
sgRNA quantification by in house script
Gene level quantification by edgeR's camera function
Supplementary files format and content: Raw counts of guide RNAs were tabulated and combined in Microsoft Excel spreadsheets.

molecule: genomic DNA

extract_protocol:
No extraction
Library construction protocol was followed per MSKCC's IGO core facility.
CRISPR screen

value: Human PSC derived neurons

value: WT

tag: treatment

value: Day 25 DOX+

tag: library

value: Targeted (32)


title: Small screen on Day 25 Dox-

value: Day 25 DOX-


title: 237-09 Plate1 NK 6


title: Small screen on Day 16 Dox-

value: Day 16 DOX-


title: SedSeq~tg_19~L31~CHX~BY4741~30C~10min~mock~BR1~Pellet

value: yeast cells


title: MNG337


title: Large screen on Day 25 Dox+ in graft

description: 220929 large pool gRNA raw and processed.xlsx

value: Day 25 Graft

value: Large (538)


title: 237-09 Plate1 NK 5


title: Large screen on Day 25 Dox+


title: MNG336


title: Large screen on Day 25 Dox-


title: 237-09 Plate1 NK 4


title: Large screen on Day 16 Dox-


title: SedSeq~tg_19~L33~CHX~BY4741~30C~10min~CHX~BR1~Total


title: 237-09 Plate1 NK 8


title: MNG335


title: 237-09 Plate1 NK 3


title: Mouse6_LarynxEpithelialCell_BioRep3

city: San Francisc

first: David

last: Julius

state: CA

institute: UCSF

data_processing:
Quality was checked with fastqc and adapter/low quality reads were trimmed with Cutadapt via TrimGalore
Assembly: mm10 containing exogenous sequence for tdTomato
Supplementary files format and content: Reads were aligned to reference genome using StarAlign
Supplementary files format and content: Processed data file is a normalized read count for each sample, which was generated using rsem-calculate-expression function of RSEM-1.3.3 in Rstudio

organism: Mus musculus

source_name: Larynx

extract_protocol:
Cells were sorted directed into Takara Smart Seq lysis buffer and then flash frozen. See Seeholzer and Julius 2024 for further details.
We performed cDNA synthesis with Smart-Seq v4 kit according to the protocol recommendatioons (Takara Bio, 634890). cDNA library was generated by using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024).  See Seeholzer and Julius 2024 for further details.

value: Larynx

value: EpCam+/tdTomto- Cell

value: tamoxifen


title: MNG334


title: 237-09 Plate1 NK 2


title: Mouse5_LarynxNE_BioRep2

value: tdTomato+ Cell


title: MNG333

value: brain


title: Mouse5_LarynxEpithelialCell_BioRep2


title: Mouse6_LarynxNE_BioRep3


title: 237-09 Plate1 NK 22


title: Small screen on Day 25 Dox+ in graft


title: Mouse4_LarynxNE_BioRep1


title: Mouse4_LarynxEpithelialCell_BioRep1


title: MNG332

value: Meningioma tumor


title: Mouse5_TracheaEpithelialCell_BioRep2

source_name: Trachea

value: Trachea


title: Mouse4_TracheaEpithelialCell_BioRep1


title: 237-09 Plate1 NK 21

value: NK


title: Mouse6_UpperTracheaNE_BioRep3


title: 237-09 Plate1 NK 1


title: Mouse5_UpperTracheaNE_BioRep2


title: MNG330


title: Mouse6_LowerTracheaNE_BioRep3


title: MNG329


title: 237-09 Plate1 NK 19


title: 237-09 Plate1 NK 18


title: Mouse5_LowerTracheaNE_BioRep2


title: MNG328


title: IGF113106

city: London

first: Paul

middle: M

last: Matthews

address: White City

country: United Kingdom

institute: Imperial College London

description: 10X genomics

data_processing:
Locally generated snRNASeq data were pre-processed using 10X Genomics Cell Ranger. Illumina sequencing files were aligned to the genomic sequence (introns and exons) using GRCh38 annotation in Cell Ranger v3.1. Nuclei were identified above background by the Cell Ranger software.
Assembly: GRCh38
Supplementary files format and content: count data and associated metadata

molecule: nuclear RNA

source_name: Brain

extract_protocol:
Single nucleus isolation was conducted on 49 tissue blocks from (n=24, NDC; n=25, AD). Homologous fresh frozen brain tissue blocks from the EC, MTG and SSC were cryosectioned at 80um and 200mg of grey matter was collected in RNAse-free Eppendorf tube. All steps were carried out on ice or at 4°C. Tissue was homogenised in buffer (1% Triton X-100, 0.4 U/μl RNAseIn + 0.2 U/μl SUPERaseIn, 1ul 1mg/ml DAPI) using a 2ml glass douncer. The homogenate was centrifuged at 4°C for 8mins, 500g and supernatant removed. The pellet then was resuspended in homogenisation buffer and filtered through a 70um filter followed by density gradient centrifugation at 13,000g for 40mins. The supernatant was removed and nuclei were washed and filtered in PBS buffer (PBS + 0.5mg/ml BSA + 0.4 U/μl RNAseIn + 0.2 U/μl SUPERaseIn). Nuclei were pelleted, washed twice in PBS buffer and resuspended in 1ml PBS buffer. 100ul of nuclei solution was set aside on ice for single nuclear processing.
Isolated nuclei stained with Acridine Orange dye were counted on a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems, L20001). Approximately 7000 nuclei were used for 10x Genomics Chromium Single Cell 3ʹ processing and library generation. All steps were conducted according to the 10x Genomics Chromium Single Cell 3' Reagent Kits v3 User Guide, with 8 cycles of cDNA amplification until fragmentation, where 25ng of amplified cDNA per sample was taken through for fragmentation. The final index PCR was conducted at 14 cycles. cDNA and library prep concentration were measured using Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851) and DNA and library preparations were assessed using the Bioanalyzer High-Sensitivity DNA Kit (Agilent, 5067-4627). Pooled samples at equimolar concentrations were sequenced on an Illumina HiSeq 4000 according to the standard 10X Genomics protocol.
snRNA-seq

value: Brain

tag: braak


title: Mouse4_LowerTracheaNE_BioRep1


title: IGF113107

description: 10X genomics


title: IGF113101

description: 10X genomics


title: MNG327


title: IGF113103

description: 10X genomics


title: IGF113102

description: 10X genomics


title: 237-09 Plate1 NK 16


title: IGF113104

description: 10X genomics


title: IGF113105

description: 10X genomics


title: MNG325


title: IGF113100

description: 10X genomics


title: 237-09 Plate1 NK 15


title: IGF113097

description: 10X genomics


title: MNG331


title: 237-09 Plate1 NK 14


title: IGF113098

description: 10X genomics


title: 237-09 Plate1 NK 17


title: IGF113099

description: 10X genomics


title: 237-09 Plate1 NK 13


title: MNG326


title: MNG323


title: 237-09 Plate1 NK 12


title: MNG324


title: IGF113096

description: 10X genomics


title: MNG322


title: IGF113057

description: 10X genomics


title: IGF113070

description: 10X genomics


title: 237-09 Plate1 NK 10


title: IGF113064

description: 10X genomics


title: IGF113068

description: 10X genomics


title: IGF113077

description: 10X genomics


title: MNG321


title: MNG320


title: IGF113056

description: 10X genomics


title: MNG319


title: IGF113076

description: 10X genomics


title: IGF113065

description: 10X genomics


title: 237-09 Plate1 NKB 8

value: NKB


title: MNG318


title: 237-09 Plate1 NKB 9


title: IGF113091

description: 10X genomics


title: MNG317


title: Vero csRNAinput-seq rep1

city: La Jolla

first: Christopher

last: Benner

state: California

address: 9500 Gilman Dr. MC 0640

department: Medicine

institute: University of California, San Diego (UCSD)

description:
short RNAs (20-65nt)
tss.Vero.bed.gz

data_processing:
csRNA-seq (small capped RNAs, ~20-60 nt) and csRNAinput-seq reads were trimmed of their adapter sequences using HOMER (“homerTools trim -3 AGATCGGAAGAGCACACGTCT -mis 2 -minMatchLength 4 -min 20”).  Reads for all genome-based assays (i.e. everything except TSS-MPRA) were aligned to the appropriate genome (GRCh38/hg38, GRCm38/mm10, ChlSab1.1) using STAR version 2.7.9a with default parameters. Only reads with a single, unique alignment (MAPQ >=10) were considered in the downstream analysis.  In the case of mouse SPRET BMDM csRNA-seq, these data were analyzed in the context of C57Bl6 BMDM csRNA-seq previously published as part of GSE135498 [PMID:31649059]. To compare how TSS change between the two strains of mice, only TSS positions that were homologous between C57Bl6 and SPRET genomes and were uniquely alignable in both genomes were considered for downstream analysis (additional details available in the manuscript)
To analyze TSS positions, csRNA/csRNAinput-seq reads with spliced or soft clipped alignments were first discarded to ensure accurate TSS assignments. The alignment position of the 5’ nucleotide of each csRNA-seq read was used to create a map of putative TSS locations in the genome for each sample set. To ensure we use high quality TSS that could be reliably quantified across different conditions, only TSS locations with at least 7 reads per 10^7 total aligned reads across all compared replicates and conditions (e.g. Ctrl siRNA, NRF1 siRNA, etc.) were kept for further analysis. Furthermore, any TSS position that had higher normalized read density in the small csRNAinput-seq data was discarded as a likely false positive TSS location. These sites often include miRNAs and other high abundance RNA species which are not entirely depleted in the csRNA-seq cap enrichment protocol. To quantify the change in TSS levels between conditions, a unified map of confident TSS positions is first determined across the set of compared experiment as described above. Then, the TSS activity levels are assessed for each replicate and each experimental condition by first counting the raw read coverage and normalizing the dataset using DESeq2’s rlog variance stabilizing transform [PMID: 25516281]. Changes in transcriptional activity were then reported as the log2 fold change representing the difference between averaged rlog transformed activity levels across conditions (similar to a shrunken fold change).
ChIP-seq peaks were found using HOMER's findPeaks program using "-style factor".
TSS-MPRA anlysis: Raw sequencing reads were trimmed for the 5’ adapter sequence GGTAACCGGTCCAGCTCA on the R1 read using cutadapt version 3.4. The trimmed reads were aligned to the reporter library using STAR version 2.7.9a, specifying an option to preclude soft-clipping at the 5’ end of R1 (STAR --outSAMattributes All --genomeDir library/tfsweep.STARIndex --runThreadN 12 --readFilesIn file.fastq --outFileNamePrefix star/Sweep_1. --alignEndsType Extend5pOfRead1 --outSAMtype BAM SortedByCoordinate). For DNA plasmid control samples, the uniquely aligned read pairs were counted to later scale transcriptional output. For RNA samples, uniquely aligned read pairs were further processed to identify exact transcription start sites, yielding a matrix of start sites per sequence position. Any alignments showing mismatches at their 5’ ends were not counted and reporter sequences with fewer than 50 total DNA alignments were also ignored. A scaling factor for each sequence was determined by calculating min(10000/plasmid, 100). After multiplying the start site counts by the scaling factor, a pseudocount of 1 was added and values were log2 transformed.
Assembly: hg38, mm10, ChlSab1.1
Supplementary files format and content: bed files: Locations indicate TSS or ChIP-seq peak positions. Numerical values associated with the files represent normalized read densities or log2 ratios between two conditions as indicated by the file names.  fa files: FASTA files containing the designed insert sequences used for TSS-MPRA experiments  tsv files: tab-delimited text files containing the TSS activity levels for each TSS-MPRA insert. Each column represents the TSS position starting within the insert starting from the beginning of the variable sequence region.  fpkm.txt file: tab-delimited text file containing RNA-seq FPKM expression values for GENCODE annotated genes.

organism: Chlorocebus sabaeus

source_name: small RNA mapping in Vero cells

growth_protocol: U2OS cells were grown at 37°C in DMEM (Cellgro) supplemented with 10% FBS (Gibco), 50 U Penicillin and 50 μg Streptomycin per mL (Gibco). For siRNA transfection, cells were washed once with PBS (Gibco), trypsinized for about 5 minutes and then washed twice with PBS (Gibco) by centrifugation at 400 g for 5 minutes. Subsequently, about 3 million cells were resuspended in 150 µl Gene Pulser® Electroporation Buffer (Biorad) and siRNAs in 1x siRNA buffer (60 mM KCl, 6 mM Hepes 7.5, 0.2 mM MgCl2). The following siRNAs were used: M-011796-02- 0005 siGENOME Human YY1 (7528) siRNA, M-017924-01- 0005 siGENOME Human NRF1 (4899) siRNA, and siGFP (CCACUACCUGAGCACCCAGU, (Meade et al. 2014)) as control. For transfection of the dominant-negative NRF1 mutant (AA1-304 (Gugneja, Virbasius, and Scarpulla 1996)), sequence was cloned into pGEM-T and mRNA synthesized using the mMESSAGE mMACHINE™ T7 Transcription Kit (Thermo Fisher Scientific). siRNAs and dnNRF1 were used at a final concentration of 10 μM. Mixture was transferred to a 0.2 cm cuvette and pulsed once at 250 V for a constant 20 msec. Following electroporation, 1 ml complete growth media was added and cells grown for 24h in a 6 cm dish.   Bone marrow derived macrophages (BMDMs) from SPRET mice were generated and treated with KLA as described in Link et. al (2018) [PMID:29779944].  TSS-MPRA experiments in HEK293T cells: MPRA inserts sequences were ordered as 200 bp oligos from Twist Bioscience and cloned Gibson-assembled into a BsaI-linearized pTSS-MPRA-Empty plasmid based on the background-reduced pGL4.10 reporter backbone (Promega), with a cloning site harboring the 18-bp pMPRA1-LH sequence followed by tandem BsaI sites for linearization and the TruSeq Read 2-compatible pMPRA1-RH sequence and a downstream landing site for reverse transcription primer RS2, and an EGFP ORF replacing the Luciferase 2 gene. About 800k HEK293T cells were used and 25 µg plasmid DNA was transfected using Lipofectamine® 3000 (Thermo Fisher Scientific) as described by the manufacturer. After 8 h, cells were washed 3x with PBS (Gibco) and RNA extracted using 1 ml Trizol (Thermo Fisher Scientific).

extract_protocol:
For all csRNA, RNA-seq, and TSS-MPRA experiments, to harvest RNA, plates were washed three times using ice cold PBS and 1 ml TRIzol™ Reagent added. Cells were scraped and RNA extracted as described by the manufacturer with addition of 1 μl 15 mg/ml GlycoBlue™ Coprecipitant (Thermo Fisher Scientific).  For ChIP-seq, cells were fixed for 10 minutes with 1% formaldehyde/PBS, and the extracted chromatin was sheared to an average DNA size of 300–500 bp.
csRNA-seq was performed as described in (Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-2 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5’-capped RNAs. Monophosphorylated RNAs were selectively degraded by 1 hour incubation with Terminator 5´-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5’dephosporylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. small RNA input (csRNAinput-seq) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 14 cycles and sequenced SE75 on the Illumina NextSeq 500.  Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq kit Stranded Total RNA Library kit (Illumina) and sequenced PE100 on Illumina NovaSeq 6000.  For ChIP-seq, one percent of the lysate was kept as ChIP input, and dnNFR1-HA was immunoprecipiated with 2 μg anti-HA antibody (Abcam ab9110) and 20 μl of Dynabeads Protein A. Libraries were prepared directly on the antibody/chromatin-bound beads using NEBNext Ultra II reagents according to the manufacturer’s protocol. Libraries were sequenced single-end for 75 cycles (SE75) to a depth of 20-25 million reads on an Illumina NextSeq 500 instrument.  For TSS-MPRA, starting with 10-15 µg RNA (⅓ of the total), non-capped RNAs were dephosphorylated with Quick CIP (NEB) and DNA digested. Samples were mixed well and incubated at 37°C for 90 minutes. For more complete dephosphorylation of uncapped transcripts, RNA was denatured a second time at 75°C for 30 seconds in a pre-warmed water bath and then quickly chilled on ice for 2 minutes before incubating the sample at 37°C for another 30 minutes. As Quick CIP is thermolabile, the brief heat treatment leaves the enzyme active. Following Cap-enrichment, RNA was purified and then vortexed hard and subsequently spun for 10 minutes at 12k g at RT. Following phase separation, the upper layer was taken, and RNA precipitated. RNA pellets were then resuspended and 5' caps removed. Following decapping, 5’ adapters were ligated by T4RNA Ligase 1 and RNA extracted. RNA pellets were then resuspended and reverse transcribed. Samples were amplified and sequenced paired-end on a NextSeq 500 (additional details available in the manuscript)

value: Vero cell line

tag: assay

value: small RNA-seq (control for csRNA-seq)

tag: genetic perturbation

tag: mouse strain


title: 237-09 Plate1 NKB 6


title: 237-09 Plate1 NKB 7


title: MNG316


title: Vero csRNA-seq rep1

description:
short RNAs (20-65nt)
tss.Vero.bed.gz

source_name: TSS mapping in Vero cells

value: csRNA-seq (transcription initiation)


title: hMG-TREM2-1w-4

city: Leuven

first: Ivana

last: Geric

address: O&N4 Herestraat 49,

country: Belgium

institute: KU Leuven, Department of Neurosciences

data_processing:
Sequencing data were mapped using STAR version=2.7.10 against the joined reference library by combining the Mus musculus reference genome (mm10/GRCm38) and the human genome (GRCh38). The full count matrix was produced by FeatureCount v1.6.3 from the Subread package.
Assembly: Mus musculus reference genome (mm10/GRCm38) and the human genome (GRCh38)
Supplementary files format and content: count matrx created by FeatureCount.

organism:
Homo sapiens
Mus musculus

extract_protocol:
RNA was isolated using the Qiagen Rneasy 96 Kit (REF 74181).
BGI BV01 library prep was used.

value: H9 (WiCell Reasearch Institute WA09)

value: microglia

value: Rag2-/- IL2r<gamma>-/- hCSF1KI APPNLGF

tag: duration

value: 1 week


title: Mouse4_UpperTracheaNE_BioRep1


title: IGF113093

description: 10X genomics


title: IGF113089

description: 10X genomics


title: IGF113087

description: 10X genomics


title: hMG-vehicle-1w-4

value: vehicle


title: MNG314


title: 237-09 Plate1 NKB 41


title: MNG313


title: IGF113082

description: 10X genomics


title: 237-09 Plate1 NKB 40


title: IGF113085

description: 10X genomics


title: MNG312


title: 237-09 Plate1 NKB 5


title: hMG-vehicle-1w-3


title: hMG-APOE-1w-4

value: APOE-ASO


title: IGF113081

description: 10X genomics


title: IGF113069

description: 10X genomics


title: 237-09 Plate1 NKB 3


title: hMG-APOE-1w-3


title: hMG-APOE-4w-4

value: 4 weeks


title: 237-09 Plate1 NKB 4


title: MNG315


title: MNG311


title: hMG-TREM2-1w-3


title: hMG-APOE-4w-3


title: IGF113086

description: 10X genomics


title: MNG310


title: 237-09 Plate1 NKB 38


title: 237-09 Plate1 NKB 39


title: MNG309


title: 237-09 Plate1 NKB 37


title: IGF113066

description: 10X genomics


title: 237-09 Plate1 NKB 43


title: MNG308


title: hMG-vehicle-4w-4


title: 237-09 Plate1 NKB 36


title: MNG307


title: hMG-vehicle-4w-3


title: 237-09 Plate1 NKB 35


title: IGF113078

description: 10X genomics


title: 237-09 Plate1 NKB 34


title: MNG305


title: 237-09 Plate1 NKB 31


title: hMG-TREM2-4w-3


title: MNG304


title: 237-09 Plate1 NKB 30


title: MNG303


title: IGF113061

description: 10X genomics


title: hMG-TREM2-4w-2


title: hMG-TREM2-4w-4


title: hMG-APOE-4w-2


title: MNG302


title: 237-09 Plate1 NKB 29


title: IGF113059

description: 10X genomics


title: hMG-APOE-4w-1


title: hMG-vehicle-4w-2


title: 237-09 Plate1 NKB 27


title: MNG301


title: IGF113083

description: 10X genomics


title: IGF113062

description: 10X genomics


title: MNG306


title: IGF113072

description: 10X genomics


title: hMG-TREM2-4w-1


title: MNG299


title: hMG-vehicle-4w-1


title: IGF113063

description: 10X genomics


title: 237-09 Plate1 NKB 25


title: IGF113058

description: 10X genomics


title: 237-09 Plate1 NKB 2


title: MNG298


title: 237-09 Plate1 NKB 24


title: hMG-vehicle-1w-2


title: IGF113094

description: 10X genomics


title: hMG-TREM2-1w-2


title: IGF113079

description: 10X genomics


title: 237-09 Plate1 NKB 23


title: hMG-APOE-1w-2


title: MNG296


title: 237-09 Plate1 NKB 20


title: IGF113067

description: 10X genomics


title: MNG295


title: MNG297


title: 237-09 Plate1 NKB 1


title: MNG300


title: hMG-APOE-1w-1


title: IGF113080

description: 10X genomics


title: 237-09 Plate1 NKB 26


title: hMG-TREM2-1w-1


title: 237-09 Plate1 NKB 19


title: MNG294


title: MNG293


title: hMG-vehicle-1w-1


title: IGF113060

description: 10X genomics


title: MNG292


title: IGF113088

description: 10X genomics


title: 237-09 Plate1 NKB 17


title: IGF113090

description: 10X genomics


title: MNG291


title: 237-09 Plate1 NKB 16


title: IGF113055

description: 10X genomics


title: IGF113075

description: 10X genomics


title: 237-09 Plate1 NKB 15


title: A549 cells infected with ZIKV sfRNAnull replicate 1 Ribo-seq

city: Ciudad de Buenos Aires

first: Horacio Martín

last: Pallarés

address: Av patricias argentinas 435

country: Argentina

institute: Fundación instituto leloir

data_processing:
RiboSeeker 0.0.1.0
Adapter sequence was trimmed using FASTX Toolkit (0.0.14), and we only keep reads with length between 20bp and 40bp
We map the filtered reads to a set of ncRNAs using bowtie2 (2.4.2)
We take the reads that are not mapped to ncRNAs and further align them to the reference genome using STAR (2.7.3a)
Genomic feature stats (CDS, UTR, intron, intergenic, and ribosome) are generated using Picard tools (2.25.7)
Assembly: hg38
Supplementary files format and content: Tab-separated values. Total raw read and labeled read counts for all  samples including read counts mapped to viral genome (MT636065)

source_name: A549 cells

growth_protocol: ZIKV infections were conducted using A549 cells (human epithelial lung cell line). A549 cells were cultured in Dulbecco's modified Eagle's medium with nutrient mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.

extract_protocol:
In brief, ribosome protected fragments were collected from duplicate samples of A549 cells infected with ZIKV wild type or sFRNAnull mutant at 20 hpi. A p100 dish of A549 cells was incubated with cycloheximide (0.1 μg/ml) in PBS for 5 minutes. The PBS was removed, and the cells were treated with Lysis buffer (20 mM total Tris HCL pH 8.0; 140 mM KCl; 1.5 mM MgCl2; 100 μg/ml Cycloheximide; 1% Triton; 40 U/ml SUPERas In (Ambion #AM2694); 10 μl/ml Protease Inhibitor Cocktail (Calbiochem #535140-1ML)). The mix was clarified for 10 minutes at 10,000g at 4°C. RNase I (100 Units, Ambion #AM2295) and DNase (10 Units, Ambion #AM2238) were added to the supernatant and incubated at room temperature for 45 minutes with gentle mixing. Digested extracts (50 μl) were then overlaid onto s-40 columns following the manufacturer’s instructions. RNA was extracted using Trizol (Invitrogen, #800868). RNA for each sample for RNA-seq was collected in parallel with the Ribosome profile experiment for RNA preparation. RNA was extracted using Trizol (Invitrogen, #800868).
Ribosome protected fragments (RPF) were separately excised in a 15% urea gel from 28-to-38 nucleotides. RNA was eluted overnight in 300 mM NaOAc pH 5.5; 1 mM EDTA; 0.1U/μl SUPERas In (Ambion #AM2694), followed by ethanol precipitation. RFP were 3’-dephosphorylated with polynucleotide kinase (10 U, PNK, New England Biolabs) in T4 polynucleotide kinase buffer (without ATP) for 1 h at 37°C, followed by a 10 min incubation at 75°C and ethanol precipitation. All RNA samples were resuspended and ligated to 3’ adaptor (30 μM /5rApp/ATCTCGTATGCCGTCTTCTGCTTG/3ddC/) using T4 RNA Ligase 2, truncated K227Q (2 U, New England Biolabs; #M0351S) and RNAseOUT Recombinant Ribonuclease Inhibitor (5 U, Invitrogen, #10777-019) for 6 h at 20°C. Ligation reactions were separately in a 10% urea gel, and fragments from 50-to-65 nucleotides for the RPF and 50-to-75 nucleotides were excised, eluted, and precipitated from the gel. The 3’-ligation products were then 5’-phosphorylated with polynucleotide kinase (15 U, PNK, New England Biolabs) in T4 polynucleotide kinase buffer supplemented with 1 mM ATP for 30 minutes at 37°C followed by RNA precipitation in ethanol. The 5’-ligation was performed using T4 RNA Ligase 1 (ssRNA Ligase, 10 U New England Biolabs, #M0204S) for 6 hrs at 20°C with barcoded 5´-Adaptor (100 μM, GUUCAGAGUUCUACAGUCCGACGAUCAAAC; GUUCAGAGUUCUACAGUCCGACGAUCGGGC; GUUCAGAGUUCUACAGUCCGACGAUCUUUC; GUUCAGAGUUCUACAGUCCGACGAUCCCCC; GUUCAGAGUUCUACAGUCCGACGAUCACUC; GUUCAGAGUUCUACAGUCCGACGAUCGACC; GUUCAGAGUUCUACAGUCCGACGAUCUGAC; GUUCAGAGUUCUACAGUCCGACGAUCCUGC). The 5’-ligation reactions were separately in a 10% urea gel, and the region from 80-to-95 nucleotides for the RPFs and 80-to-105 nucleotides for the RNA input samples was excised, eluted, and precipitated from the gel. Gel-purified samples were reverse transcribed using SuperScript™ II Reverse Transcriptase (Invitrogen) with RTOligo (CAAGCAGAAGACGGCATACGA) according to the manufacturer (Invitrogen). Amplification was done using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) and oligos: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA and CAAGCAGAAGACGGCATACGA. PCR was carried out with an initial 30 s denaturation at 98°C, followed by 4 cycles of 30 s denaturation at 98°C, and 15 s extension at 72°C. Followed by 10- 15 cycles of 10 s denaturation at 98°C, 30 s annealing at 50°C, and 15 s extension at 72°C. Several reactions, with different numbers of cycles, were performed. Reactions were separated on a non-denaturing 8% polyacrylamide TBE gel, and DNA fragments of the correct size were extracted and sequenced using Illumina HiSeq2000.

treatment_protocol: Infection with ZIKV WT and ZIKV sfRNAnull

value: A549 cells

value: lung carcinoma epithelial cells

value: WT

value: ZIKV sfRNAnull infected


title: A549 cells infected with ZIKV WT replicate 1 Ribo-seq

value: ZIKV WT infected


title: A549 cells infected with ZIKV WT replicate 2 Ribo-seq


title: MNG289

value: brain

value: Meningioma tumor


title: 237-09 Plate1 NKB 18

value: NKB


title: MNG290


title: IGF113073

description: 10X genomics

value: Brain


title: MNG288


title: A549 cells mock infected replicate 1 Ribo-seq

value: mock infected


title: MNG287


title: 237-09 Plate1 NKB 13


title: A549 cells mock infected replicate 2 Ribo-seq


title: 237-09 Plate1 NKB 14


title: A549 cells infected with ZIKV sfRNAnull replicate 2 Ribo-seq


title: 237-09 Plate1 NKB 12


title: WGBS_MECP2_WT_rep1

city: Cambridge

first: Yi

last: Liu

state: MA

address: 455 Main St

institute: Whitehead Institute for Biomedical Research

description: Whole genome bisulfite sequencing of hESC derived neurons with WT MECP2

data_processing:
For WGBS data, adapters were trimmed with Trim Galore! (v0.6.7), and reads were mapped to the bisulfite-prepared canonical chromosomes of the human genome (GRCh38/hg38) with bismark (v0.22.3) and deduplicated. The methylation status of each cytosine of each read was assayed with bismark_methylation_extractor, and methylation fraction and read coverage at each position were calculated with the bedtools suite (v2.29.2). Cytosines were partitioned into CpG and mCA contexts, and any sites with read coverage less than 5 were removed. The bedtools suite was used to further mean-summarize methylation fraction across genome features.
Assembly: hg38
Supplementary files format and content: bigWig

source_name: neuron

extract_protocol: Three-week differentiated endogenous-tagged WT MECP2-GFP NGN2-neurons were collected for the genomic DNA extraction using the Quick-gDNA MicroPrep (Zymo D3020). Bisulfite conversion and library preparation were performed using Pico Methyl-Seq Library Prep Kit (Zymo D5455) according to the manufacturer’s instructions. Libraries were pooled and sequenced on NovaSeq 6000 (Illumina) on a paired-end 100x100 run with ~650-800 million reads.

value: neuron

value: hES-neurons

value: WT MECP2


title: WGBS_MECP2_WT_rep2


title: MNG286


title: WGBS_MECP2_WT_rep3


title: A549 cells infected with ZIKV sfRNAnull replicate 1 RNA-seq


title: ATAC_MECP2_WT_rep2

description: ATAC-seq of hESC derived neurons with WT MECP2

data_processing:
Using the hg38 reference genome, raw ATAC-seq reads were processed using the ENCODE ATAC-seq analysis pipeline (https://github.com/ENCODE-DCC/atac-seq-pipeline).
Assembly: hg38
Supplementary files format and content: bigWig

growth_protocol: ATAC-Seq was performed with MECP2 WT neurons using an ATAC-Seq Kit (Active Motif 53150) according to the manufacturer’s instructions.

extract_protocol: ATAC-Seq Kit (Active Motif 53150)


title: 237-09 Plate1 B 8

value: B


title: MNG284


title: ATAC_MECP2_WT_rep1


title: MNG285


title: WT_pol2_rep3

description: RNA polymerase II

data_processing:
Sequence reads were mapped to the hg38 reference genome using Bowtie 2 with the following options: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. Following the CUT&Tag analysis protocol of Zheng, Ahmad and Henikoff (https://yezhengstat.github.io/CUTTag_tutorial/), bigWig files were generated after normalization the the E. coli spike-in reads.
Assembly: hg38
Supplementary files format and content: bigWig
Library strategy: CUT&Tag

source_name: hES-neurons

growth_protocol: hESC- neurons were maintained in BrainPhys media supplemented with N2/B27 supplements, NT-3, GDNF, and BDNF in humidified atmosphere with 5% CO2 at 37C

extract_protocol: CUT and Tag-IT kit from Active Motif.

value: MECP2 WT


title: MNG283


title: A549 cells infected with ZIKV WT replicate 2 RNA-seq


title: 237-09 Plate1 B 6


title: 237-09 Plate1 B 5


title: A549 cells mock infected replicate 2 RNA-seq


title: 237-09 Plate1 B 7


title: WT_pol2_rep2

description: RNA polymerase II


title: MNG282


title: MNG280


title: WT_pol2_rep1

description: RNA polymerase II


title: 237-09 Plate1 B 4


title: MNG281


title: CLL, SpiD3 2uM, rep3

city: Omaha

first: Jordan

last: Rowley

state: NE

address: 3828 Jones St, Apt 202

institute: University of Nebraska Medical Center

data_processing:
For each sample, quality control was assessed by the Fragment Analyzer Automated CE System.
Sequenced reads were mapped using HISAT2 version 2.1.0 to genome build hg38, guided by ENSEMBL GRCh38.99 transcript annotations.
TPM and FPKM normalized values were obtained by Stringtie version 2.1.1.
Assembly: hg38
Supplementary files format and content: Matrix table with raw gene counts and expression values for every gene and every sample

source_name: B-cell

growth_protocol: OSU-CLL cell lines were cultured in RPMI-1640 with 2 mM L-glutamine, supplemented with 100 U/mL penicillin/100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum

extract_protocol:
OSU-CLL cells (1.5e6 cells/mL) were collected for RNA extraction using the miRNeasy Mini Kit (Qiagen) according to manufacturer instructions
Samples were prepared using the Universal Plus mRNA-Seq with NuQuant kit

treatment_protocol: OSU-CLL cells were treated with SpiD3 (1, 2 μM) or DMSO for 4 h

value: B-cell

value: OSU-CLL

value: CLL

value: 2 uM SpiD3


title: 237-09 Plate1 B 3


title: WT_IgG_rep4

description: IgG


title: A549 cells infected with ZIKV sfRNAnull replicate 2 RNA-seq

value: A549 cells

value: lung carcinoma epithelial cells

value: WT


title: A549 cells mock infected replicate 1 RNA-seq


title: WT_pol2_clone2_rep1

description: RNA polymerase II


title: MNG279

value: brain


title: 237-09 Plate1 B 9


title: CLL, SpiD3 1uM, rep3

value: 1 uM SpiD3


title: 237-09 Plate1 B 2


title: MNG278

value: Meningioma tumor


title: WT_IgG_rep3

description: IgG


title: A549 cells infected with ZIKV WT replicate 1 RNA-seq

value: ZIKV WT infected


title: CLL, vehicle, rep3

value: Vehicle


title: 237-09 Plate1 B 20


title: MNG277


title: CLL, SpiD3 2uM, rep2


title: WT_IgG_rep1

description: IgG


title: 237-09 Plate1 B 1


title: CLL, SpiD3 1uM, rep2


title: MNG276


title: WT_H3K9me3_rep4


title: WT_H3K9me3_rep3


title: MNG274


title: CLL, vehicle, rep2


title: CLL, SpiD3 2uM, rep1


title: 237-09 Plate1 B 17


title: MNG275


title: MNG273


title: WT_H3K9me3_rep2


title: 237-09 Plate1 B 16


title: CLL, SpiD3 1uM, rep1


title: MNG272


title: CLL, vehicle, rep1


title: WT_IgG_rep2

description: IgG


title: MNG271


title: 237-09 Plate1 B 15


title: 237-09 Plate1 B 14


title: WT_H3K4me3_rep3


title: 237-09 Plate1 B 19


title: MNG270


title: 237-09 Plate1 B 13


title: WT_H3K4me3_rep1


title: 44hAPF Brain ATAC Rep3

city: Ann Arbor

first: Elizabeth

last: Fogarty

state: MI

address: 1105 N. University Ave

institute: University of Michigan

data_processing:
Adaptors and low-quality bases trimmed with cutadapt version 1.18, -q 20, -m 25
Alignment to Drosophila genome dm6 with bowtie2 version 2.4.1, --local --very-sensitive -X 1000
PCR duplicates marked with Picard version 2.8.1 MarkDuplicates
Bam file generation, sub-nucleosomal fragments selected, downsampling, sorting, and indexing with samtools version 1.5
Bigwig file generation with Deeptools bamCoverage --binsize 10 --extendreads --normalizeUsing CPM --ignoreForNormalization chrM
Peaks called using py-macs2 verison 2.2.4 callpeak
Read coverage in peaks quantified by subread version 2.0.3 featurecounts
Assembly: dm6
Supplementary files format and content: BigWig

organism: Drosophila melanogaster

growth_protocol: Animals were raised at 25°C on Bloomington Cornmeal media without malt extract

extract_protocol:
Dissect 16 eyes or 3 brains per sample in Wash+ solution (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM Spermidine and Roche complete Protease Inhibitor Tablet: 1 tablet per 50 mL). Pipet 100ul of dissected tissues in Wash+ solution directly into 100µL 2X Collagenase/Dispase solution. Incubate at 23C for 30 minutes, shaking at 500 rpm. Vortex for 10 seconds at 60% max speed on a benchtop vortexer. Incubate another 10 minutes at 23C, shaking at 500 rpm. Vortex again for 10 seconds at 60% speed. From here, we use the Omni-ATAC Protocol (Corces et al., 2017). Spin down at 800 x g, 5 minutes, at 4C. Remove supernatant and wash in 200 µL 1X PBS. Repeat 4C spin and remove supernatant. Resuspend cell pellet in 50 µl cold ATAC-RSB (10mM Tris-HCl pH 7.4, 10 mM NaCl, 3mM MgCl2) supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. Pipette up and down 3 times. Incubate on ice 3 minutes. Add 1 mL cold ATAC-RSB containing 0.1% Tween-20 without NP40 or Digitonin. Invert tube 3 times. Spin down at 800 x g for 10 minutes, 4°C. Discard supernatant and resuspend nuclei in 50 µL transposition reaction mix: 25 µl 2x TDE1 Buffer, 3.5 µl Tn5 Enzyme, 16.5 µl PBS, 0.5 µL 1% Digitonin, 0.5 µL 10% Tween-20, 5 µL water. Incubate at 37°C for 30 minutes shaking at 1000 rpm.Purify using a Qiagen MinElute kit according to manufacturer’s protocol. Elute in 21 µl Elution Buffer (10 mM Tris buffer pH 8.0).
Library amplification was performed with NEBNext High-Fidelity 2X PCR Master Mix, according to cycling conditions described in Corces et al., 2017, and qPCR side reactions to optimize the number of PCR cycles for each sample. Libraries were assessed on an Agilent Bioanalyzer prior to sequencing

treatment_protocol: Larval samples were dissected from wandering larvae isolated from uncrowded vials. Vials with more than ~100 larvae were diluted into fresh vials to keep larvae uncrowded. Pupa were collected from vials at the White Pre-pupa stage (WPP) as described (Flegel et al., 2013), which was taken as 0h After Puparium Formation (APF) and reared on damp Kimwipes at 25°C to the indicated hours APF.

tag: Sex

value: female

value: y,w,hsflp/w ; + ; +


title: MNG269


title: 44hAPF Brain ATAC Rep2


title: 44hAPF Brain ATAC Rep1


title: 237-09 Plate1 B 18

data_processing:
The fastq files were aligned to MacaM v7 using STAR aligner v 2.5.2b. The raw counts from ReadsPerGene output of STAR was used for downstream analysis using Seurat v4.0.4.
Assembly: MacaM v7
Supplementary files format and content: The ReadsPerGene output from STAR for each cell was combined as separate columns in ReadCounts.txt.The MacaM v7.8.2 gtf was used to convert gene id to gene names.


title: MNG268


title: WT_GFP_rep3

description: WT MECP2 (anti-GFP)

value: hES-neurons


title: WT_H3K9me3_rep1


title: 24hAPF Brain ATAC Rep3


title: 94-09 Plate0 NKB H9

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H9

value: NKB


title: MNG267


title: 94-09 Plate0 NKB H8

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H8


title: 24hAPF Brain ATAC Rep2


title: MNG266


title: 94-09 Plate0 NKB H7

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H7


title: 24hAPF Brain ATAC Rep1


title: WT_H3K4me3_rep2

value: MECP2 WT


title: MNG265


title: WT_GFP_clone2_rep1

description: WT MECP2 (anti-GFP)


title: 94-09 Plate0 NKB H6

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H6


title: IGF113092

description: 10X genomics

value: Brain


title: 237-09 Plate1 B 12

value: B


title: 94-09 Plate0 NKB H5

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H5


title: MNG264


title: L3 Brain ATAC Rep2


title: L3 Brain ATAC Rep3


title: MNG263


title: 94-09 Plate0 NKB H4

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H4


title: WT_GFP_ab2_rep1


title: 44hAPF Eye ATAC Rep3

source_name: eye

value: eye


title: R270X_pol2_rep3

description: R270X (anti-GFP)

value: MECP2 R270X


title: MNG262


title: 44hAPF Eye ATAC Rep2


title: WT_GFP_rep2

description: WT MECP2 (anti-GFP)


title: MNG260


title: 94-09 Plate0 NKB H1

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H1


title: R270X_pol2_rep2

description: R270X (anti-GFP)


title: WT_GFP_rep1

description: WT MECP2 (anti-GFP)


title: MNG261


title: 94-09 Plate0 NKB H11

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H11


title: WT_GFP_ab2_rep3


title: MNG259


title: R270X_pol2_rep1

description: R270X (anti-GFP)


title: 24hAPF Eye ATAC Rep3


title: WT_GFP_ab2_rep2


title: R270X_pol2_clone2_rep1

description: R270X (anti-GFP)


title: MNG258


title: 24hAPF Eye ATAC Rep1


title: 94-09 Plate0 NKB H10

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H10


title: 94-09 Plate0 NKB G9

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G9


title: L3 Brain ATAC Rep1


title: 94-09 Plate0 NKB G8

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G8


title: MNG257


title: R270X_GFP_rep3

description: R270X (anti-GFP)


title: R270X_GFP_rep2

description: R270X (anti-GFP)


title: 24hAPF Eye ATAC Rep2


title: 94-09 Plate0 NKB H2

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_H2


title: R270X_GFP_rep1

description: R270X (anti-GFP)


title: L3 Eye ATAC Rep3

source_name: eye disc

value: eye disc


title: 94-09 Plate0 NKB G7

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G7


title: MNG255


title: MNG256


title: 44hAPF Eye ATAC Rep1


title: L3 Eye ATAC Rep2


title: L3 Eye ATAC Rep1


title: MNG254


title: R270X_GFP_clone2_rep1

description: R270X (anti-GFP)


title: R168X_pol2_rep3

description: R168X (anti-GFP)

value: MECP2 R168X


title: 94-09 Plate0 NKB G5

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G5


title: MNG253


title: 94-09 Plate0 NKB G6

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G6


title: MNG252


title: 94-09 Plate0 NKB G4

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G4


title: 94-09 Plate0 NKB G3

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G3


title: R168X_pol2_rep2

description: R168X (anti-GFP)


title: R168X_pol2_rep1

description: R168X (anti-GFP)


title: 94-09 Plate0 NKB G2

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G2


title: R168X_GFP_rep3

description: R168X (anti-GFP)


title: MNG251


title: R168X_pol2_clone2_rep1

description: R168X (anti-GFP)


title: 94-09 Plate0 NKB G1

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G1


title: MNG250


title: R168X_GFP_rep2

description: R168X (anti-GFP)


title: 44hAPF Eye RNA Rep1

data_processing:
Adaptors and low-quality bases trimmed with cutadapt version 1.18, -q 20, -m 25
Alignment to Drosophila genome dm6 with STAR version 2.5.2a
Bam file generation, sorting, and indexing with samtools version 1.5
Read coverage in genes quantified by subread version 1.6.0 featurecounts
Raw counts were normalized and Log2(CPM) values generated with EdgeR
Assembly: dm6
Supplementary files format and content: tab-delimited text file contains normalized Log2(CPM) gene counts for all samples

extract_protocol:
Dissected 16 eyes per sample in 1X PBS. Eye discs were separated from antenna discs during dissection. RNA was extracted using a standard Trizol/chloroform protocol, precipitated overnight at -20C in isopropanol, and quality was assessed with Agilent Bioanalyzer.
Libraries were prepared using NEBNext Poly(A) mRNA Stranded Library Prep Kit and assessed on an Agilent Bioanalyzer prior to sequencing

tag: age


title: MNG249


title: 94-09 Plate0 NKB G11

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G11

extract_protocol:
RNA was purified using RNACleanXP Solid Phase Reversible Immobilization (SPRI) beads (Beckman Coulter). Beads with bound RNA were re-suspended in 5 ul of RSB buffer.
Single-cell RNA sequencing was performed by Medgenome using a modified version of the ClonTech SMART-Seq Single Cell Kit that utilized miniaturization to ¼ volumes using a MANTIS Liquid Handler (Formulatrix). Amplified cDNA was fragmented and appended with dual-indexed barcodes using Illumina Nextera kits.


title: 94-09 Plate0 NKB G10

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_G10


title: 44hAPF Eye RNA Rep3


title: 24hAPF Eye RNA Rep1


title: 24hAPF Eye RNA Rep3


title: MNG248


title: 24hAPF Eye RNA Rep2


title: R168X_GFP_rep1

description: R168X (anti-GFP)


title: MNG247


title: 94-09 Plate0 NKB F9

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F9


title: MNG246


title: L3 Eye RNA Rep1


title: R168X_GFP_clone2_rep1

description: R168X (anti-GFP)


title: 44hAPF Eye RNA Rep2


title: 94-09 Plate0 NKB F8

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F8


title: L3 Eye RNA Rep2


title: R133C_pol2_rep3

description: R133C (anti-GFP)

value: MECP2 R133C


title: MNG245

value: brain


title: 94-09 Plate0 NKB F7

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F7


title: MNG244


title: KOm7oex4

city: Berlin

first: Nikolaus

last: Rajewsky

address: Hannoversche Str. 28

department: BIMSB

country: Germany

institute: Max Delbrück Center for Molecular Medicine

data_processing:
fastqc to check quality of sequencing reads
bcl2fastq (v2.20.0) to demultiplex fastq files
STAR (v2.7.0a) count to align sequencing files to a reference transcriptome
featureCounts (v1.6.0) to count mapped reads for genomic features
Assembly: mm10
Supplementary files format and content: comma-separated raw read counts in matrix.csv

source_name: Primary mouse cortical neuron, Cdr1as KO and miR-7 overexpression, DIV21

extract_protocol:
Total RNA from primary neurons was extracted using home-made TRIzol reagent in combination with Direct-zol RNA Kit (Zymo Research).
RNA libraries for RNA-seq were prepared using TruSeq (v2) library Prep Kit following manufacturer's protocols.

value: Primary mouse cortical neuron

value: Cdr1as KO

value: miR-7 overexpression


title: 94-09 Plate0 NKB F6

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F6

source_name: NKB


title: R133C_pol2_rep1

description: R133C (anti-GFP)


title: KOm7oex2


title: 94-09 Plate0 NKB F5

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F5


title: MNG242

data_processing:
RNA sequencing reads were aligned to the Gencode GRCh38.primary_assembly genome using STAR2 (v2.7.7a) and then using HTSeq (v0.11.0) reads were counted for each associated gene using the Gencode V39 primary assembly annotations
Gene expression values from combined datasets were batch corrected and normalized and converted to units of log2 transcripts per million (log2TPM)
Assembly: hg38
Supplementary files format and content: tab-delimited files contain log2 TPM values for each sample

extract_protocol:
RNA was extracted using the Qiagen RNeasy Plus mini kit. Total RNA integrity was checked using an Agilent 4200 TapeStation (Agilent Technologies, Inc., Santa Clara, CA) and quantified using a Trinean DropSense96 spectrophotometer (Caliper Life Sciences, Hopkinton, MA). Samples with RIN < 5 were removed from further analysis. RNA was normalized to 50ng/ul and 500ng was submitted for library preparation.
RNA-seq libraries were prepared from total RNA using the TruSeq Stranded mRNA kit (Illumina, Inc., San Diego, CA, USA). Sequencing was performed using an Illumina NovaSeq 6000 employing a paired-end, 50 base read length (PE50) sequencing strategy. RNA extraction was performed by the Specimen Processing & Research Cell Bank at Fred Hutch. Library preparation and sequencing was performed by the Genomics and Bioinformatics Core Services at Fred Hutch.

value: Meningioma tumor


title: KOm7oex3


title: R133C_pol2_clone2_rep1

description: R133C (anti-GFP)


title: R133C_GFP_rep3

description: R133C (anti-GFP)


title: 94-09 Plate0 NKB F3

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F3


title: MNG243


title: 94-09 Plate0 NKB F2

description: Multi.cell_sort__44NKB__22NK__22B__Spleen_from_94.09__4_6_21_F2


title: KOC3

source_name: Primary mouse cortical neuron, Cdr1as KO, DIV21


title: MNG240

source_name: brain


title: KOC4


title: R133C_GFP_rep2

description: R133C (anti-GFP)


title: KOm7oex1