title: Sample 60_Preglobular_Rep2_Technical4_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 4
Preglobular_Rep2_Technical4_MethylC-Seq


title: Sample 66_Preglobular_Rep3_Technical2_MethylC-Seq

status: Public on Jun 25 2020

type: SRA

city: Vienna

first: Ranjith

last: Papareddy

address: Dr Bohr Gasse 3

country: Austria

institute: Gregor Mendel Institute

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate3 Technical replicate 2
Preglobular_Rep3_Technical2_MethylC-Seq

library_source: genomic

data_processing:
Small RNA-seq Analysis: Adaptor trimming with Cutadapt parameters "cutadapt -a AGATCGGAAGA  -m 18 -M 30 --trimmed-only". End to End alignment with zero mismatched and upto 100 multimappers  against TAIR10  Arabidopsis thaliana genome with STAR "STAR --outFilterMultimapNmax 100 --outSAMtype SAM --outFilterMismatchNmax 0 --alignEndsType EndToEnd --outReadsUnmapped Fastx --alignIntronMax 1 --outSAMprimaryFlag AllBestScore".  Multi mapping reads rescued with rich-get-richer algorithem " readmapIO.py  ". Resulting bedfiles sorted, collapsed and normalized to totoal genome matching reads.
MethylC-Seq Analysis: Adaptor trimming with  trimeGalore (a wraper up trimmerer from Cutadapt) along with quality trimming, 6nt from 3' and 5' end were remove to exclude random hexamers used in preamplification step of methylome library preperation. After quality filtering and adaptor trimming, bisulfite-converted reads were aligned against the TAIR10 genome  using Bismark (bismark --non_directional -q --score-min L,0,-0.4) and deduplicated with function deduplicate_bismark -s --bam. Aligned BAM files with unique mapping reads and clonal deduplated were used as  input for the Methylpy software (https://bitbucket.org/schultzmattd/methylpy) to extract weighted methylation rate at each covered Cytosine site. Bisulfite conversion rates were calculated using the unmethylated chloroplast genome or spiked-in unmethylated Lambda phage DNA controls (European Nucleotide Archive Accession Number J02459, Promega catalog number D1521)
Genome_build: TAIR10
Supplementary_files_format_and_content: sRNA-Seq: Bed files contain tab-separated values of chromosomes (column 1), start (column 2) and stop (column 3) coordinates of smallRNAs followed by normalize RPM score (column 4), strand (column 5), sequence (column 6) and small RNA size (column 7)
Supplementary_files_format_and_content: MethylC-seq:  All Cytosin containing tab-separated values of chromosomes (column 1), start position(column 2)  of cytosine followed by strand  (column 3), Trinucleotide context  (column 4), count of reads supporting methylation (column 5) and total reads  (column 6); In addition ,Bigwig files containing chromosomes (column 1), start (column 2) and stop (column 3) and weighted methylation rate (column 4)

molecule: genomic DNA

organism: Arabidopsis thaliana

growth_protocol: Controlled growth chamber at 20˚C-22˚C with a 16-h light/8-h dark cycle. Plants were four weeks old when tissue was harvested.

extract_protocol:
samples 1 to 28 : Total RNA extracted with TRIzol reagent (Thermo Fisher Scientific) and size selected for 18-30nt RNA molecules with denaturing urea gel; Samples 29 to 69 : genomic DNA extracted with Quick-DNATM Micro prep Kit (Zymo D3020)
Samples 1 to 28 : Size selected RNA was used to synthesise cDNA with NEBNext Multiplex Small RNA Library Prep Set for Illumina kit (cat. no E7300; New England Biolabs) according to the manufacturer’s recommendations. Various numbers of PCR cycles were used to amplify cDNAs: 18, 20, 22, and 24 PCR cycles for globular; 14, 16, 18, and 20 PCR cycles for early heart and 3 weeks old leaf samples. Final PCR amplicons were initially run on a 90% (v/v) formamide/8% (w/v) acrylamide gel for 30 min at 5W, followed by 30W for ≥2 h, and stained with SYBR Gold (1:10,000; Thermo Fisher Scientific). Intensity of PCR amplicons between 137-bp and 147-bp  Fluorescence intensities of amplicons were examined across the PCR cycles, 137-bp to 149-bp corresponding to 18- nucleotide to 30- nucleotide sRNAs with adapters respectively was inspected under UV transilluminator. Amplicons with  non-saturated signals from PCR cycles were gel-purified. Gel purified small RNA libraries are resuspenden in 15 μL of Elution Buffer (Qiagen). Finally small RNA libraries quality checked for expected size range with Agilent High sensitivity NGS fragment Kit (DNF-474-1000) were sequenced on a HiSeq 2500 instrument (Illumina) in 50 base single-end mode.
Samples 29 to 69 : Genomic DNA subjected to genomic DNA  bisulfite treatment for 200 minutes with EZ DNA Methylation-DirectTM Kit (Zymo D5020).preamplified for two cycles with primers (5′-CCCTACACGACGCTCTTCCGATCTNNNNNN-3′) containing random hexamers and purified using the Zymo DNA Clean and Concentrator kit. Adaptor primers (5′-CAGACGTGTGCTCTTCCGATCTNNNNNN-3′) were added to preamplified products and then amplified for 12 PCR cycles with indexing primers for Illumina sequencing. Methylome libraries were purified using Beckman Coulter AMPureXP DNA beads.
samples 1 to 28 : smallRNA-seq; samples 29 to 69: MethylC-Seq

tag: genotype acronym

tag: genotype

value: wild type

tag: tissue

value: Preglobular (8/16 cell) embryo

tag: treatment nucleic acids prior to library construction

value: Bisulfite treated

tag: amount of total rna used for library construction


title: Sample 62_Preglobular_Rep2_Technical6_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 6
Preglobular_Rep2_Technical6_MethylC-Seq


title: Sample 63_Preglobular_Rep2_Technical7_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 7
Preglobular_Rep2_Technical7_MethylC-Seq


title: Sample 65_Preglobular_Rep3_Technical1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate3 Technical replicate 1
Preglobular_Rep3_Technical1_MethylC-Seq


title: Sample 64_Preglobular_Rep2_Technical8_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 8
Preglobular_Rep2_Technical8_MethylC-Seq


title: Sample 59_Preglobular_Rep2_Technical3_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 3
Preglobular_Rep2_Technical3_MethylC-Seq


title: Sample 58_Preglobular_Rep2_Technical2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 2
Preglobular_Rep2_Technical2_MethylC-Seq


title: human mRNA 1

city: Canberra

first: Susan

last: Wagner

state: ACT

address: 131 Garran Road

department: The John Curtin School of Medical Research

country: Australia

institute: Australian National University

description: Raw data were originally paired-end but only read 1 was analysed and is provided here.

library_source: transcriptomic

data_processing:
Illumina CASAVA 1.8 for base calling
adapter trimming (fastx_clipper of FASTX Toolkit 0.0.13), filtering by alignment to rRNA (bowtie 1.2.2) and ncRNA, tRNA (bowtie2 2.2.6)
alignment to transcriptome Ensembl GRCh38.80 and genome GRCh38 with TopHat 2.1.0
Genome_build: GRCh38
Supplementary_files_format_and_content: human_metagene_FP5end_AUG_-80+30_FPlength.ods; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the AUG start codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: human_metagene_FP5end_STOP_-80+30_FPlength.ods; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the STOP codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: human_metagene_coverage_CAP_-40+40.csv; footprint coverage from -40 nt to +40 nt relative to the transcript start
Supplementary_files_format_and_content: human_genewise_FPcount_transcript_area.csv; footprint count per transcript area
Supplementary_files_format_and_content: human_genewise_mean_CI_FP5end_FP3end_AUG_-50+50.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints without 5’extension (footprint 5’end from -12 to -14 nt)
Supplementary_files_format_and_content: human_coverage_ATF4_ENST00000396680_-50.csv; coverage for the 5’UTR and the beginning of the CDS for ATF4 (starting at -50 nt from the transcript start)

molecule: polyA RNA

organism: Homo sapiens

source_name: HEK293T cells

growth_protocol: 3.5 Mio cell seeded into one Ø 15 cm dish, 48 h growth, around 80 % confluency at harvest; growth at 37ºC, 5 % CO2, in DMEM + 10% FBS (20 ml per dish)

extract_protocol:
in-plate cell lysis with detergent, total RNA extraction with RNeasy Mini Kit, poly(A) selection with poly(A)Purist MAG Kit, fragmentation
according to Ingolia et al., 2010, Methods Enzymol (different RNA fragment size selection, different set of rRNA depletion oligos)

treatment_protocol: incubation of the cell culture with 0.3 % w/v formaldehyde for 5 min in a cold room

tag: antibody

value: no

tag: cell line

tag: protocol

value: RNA-seq


title: Sample 69_Preglobular_Rep3_Technical5_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate3 Technical replicate 5
Preglobular_Rep3_Technical5_MethylC-Seq


title: Sample 55_Preglobular_Rep1_Technical2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate1 Technical replicate 2
Preglobular_Rep1_Technical2_MethylC-Seq


title: Sample 51_Leaf_WT_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Leaf_WT Biological replicate2
Leaf_WT_Rep2_MethylC-Seq

value: 3-weeks old leaf


title: Sample 56_Preglobular_Rep1_Technical3_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate1 Technical replicate 3
Preglobular_Rep1_Technical3_MethylC-Seq


title: Sample 57_Preglobular_Rep2_Technical1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate2 Technical replicate 1
Preglobular_Rep2_Technical1_MethylC-Seq


title: Sample 52_Noncotyledon_portion_Rep1_HiSeq_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Noncotyledon_portion Biological replicate1_HiSeq
Noncotyledon_portion_Rep1_HiSeq_MethylC-Seq

value: Non Cotyledon_portion (8DAP) of embryo


title: Sample 49_Leaf_nrpd1a-3_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Leaf_nrpd1a-3 Biological replicate2
Leaf_nrpd1a-3_Rep2_MethylC-Seq

value: RNA Polymerase IV mutant


title: Sample 48_Leaf_nrpd1a-3_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Leaf_nrpd1a-3 Biological replicate1
Leaf_nrpd1a-3_Rep1_MethylC-Seq


title: Sample 46_FloralBud_WT_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from FloralBud_WT Biological replicate1
FloralBud_WT_Rep1_MethylC-Seq

value: Floral bud


title: Sample 50_Leaf_WT_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Leaf_WT Biological replicate1
Leaf_WT_Rep1_MethylC-Seq


title: Sample 47_FloralBud_WT_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from FloralBud_WT Biological replicate2
FloralBud_WT_Rep2_MethylC-Seq


title: Sample 43_EarlyHeart_WT_Rep3_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_WT Biological replicate3
EarlyHeart_WT_Rep3_MethylC-Seq

value: Early heart  (4DAP) embryo


title: Sample 44_FloralBud_nrpd1a-3_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from FloralBud_nrpd1a-3 Biological replicate1
FloralBud_nrpd1a-3_Rep1_MethylC-Seq


title: Sample 45_FloralBud_nrpd1a-3_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from FloralBud_nrpd1a-3 Biological replicate2
FloralBud_nrpd1a-3_Rep2_MethylC-Seq


title: Sample 42_EarlyHeart_WT_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_WT Biological replicate2
EarlyHeart_WT_Rep2_MethylC-Seq


title: Sample 54_Preglobular_Rep1_Technical1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Preglobular Biological replicate1 Technical replicate 1
Preglobular_Rep1_Technical1_MethylC-Seq


title: Sample 41_EarlyHeart_WT_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_WT Biological replicate1
EarlyHeart_WT_Rep1_MethylC-Seq


title: Sample 40_EarlyHeart_nrpd1a-3_Rep3_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_nrpd1a-3 Biological replicate3
EarlyHeart_nrpd1a-3_Rep3_MethylC-Seq


title: Sample 39_EarlyHeart_nrpd1a-3_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_nrpd1a-3 Biological replicate2
EarlyHeart_nrpd1a-3_Rep2_MethylC-Seq


title: Sample 34_BentCotyledon_WT_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_WT Biological replicate1
BentCotyledon_WT_Rep1_MethylC-Seq

value: Bent cotyledon (8DAP) embryo


title: Sample 33_BentCotyledon_nrpd1a-3_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_nrpd1a-3 Biological replicate2
BentCotyledon_nrpd1a-3_Rep2_MethylC-Seq


title: Sample 32_BentCotyledon_nrpd1a-3_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_nrpd1a-3 Biological replicate1
BentCotyledon_nrpd1a-3_Rep1_MethylC-Seq


title: Sample 35_BentCotyledon_WT_Rep2_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_WT Biological replicate2
BentCotyledon_WT_Rep2_MethylC-Seq


title: Sample 28_leaf_suvh456_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_suvh456 from replicate 3
leaf_suvh456_Rep3.sRNA-seq

molecule: total RNA

value: histone H3K9 methyltransferases SUVH4,5 and 6 triple mutant


title: Sample 30_BentCotyledon_0.5ng_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_0.5ng
BentCotyledon_0.5ng_MethylC-Seq


title: Sample 29_BentCotyledon_0.1ng_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_0.1ng
BentCotyledon_0.1ng_MethylC-Seq


title: Sample 25_leaf_nrpd1a-3_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_nrpd1a-3 from replicate 3
polIV_leaf3.sRNA-seq


title: Sample 26_leaf_suvh456_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_suvh456 from replicate 1
leaf_suvh456_Rep1.sRNA-seq


title: Sample 24_leaf_nrpd1a-3_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_nrpd1a-3 from replicate 2
polIV_leaf2.sRNA-seq


title: Sample 22_leaf_Hon12_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_Hon12 from replicate 3
leaf_Hon12_Rep3.sRNA-seq

value: Linker Histone 1 and 2 double mutant


title: Sample 23_leaf_nrpd1a-3_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_nrpd1a-3 from replicate 1
polIV_leaf1.sRNA-seq


title: Sample 21_leaf_Hon12_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_Hon12 from replicate 2
leaf_Hon12_Rep2.sRNA-seq


title: Sample 38_EarlyHeart_nrpd1a-3_Rep1_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from EarlyHeart_nrpd1a-3 Biological replicate1
EarlyHeart_nrpd1a-3_Rep1_MethylC-Seq


title: Sample 19_leaf_dcl234_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_dcl234 from replicate 3
d234_leaf3.sRNA-seq

value: dicer-like2,3 and 4 triple mutant


title: Sample 36_Cotyledon_portion_Rep1_HiSeq_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from Cotyledon_portion Biological replicate1_HiSeq
Cotyledon_portion_Rep1_HiSeq_MethylC-Seq

value: Cotyledon_portion (8DAP) of embryo


title: Sample 18_leaf_dcl234_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_dcl234 from replicate 2
d234_leaf2.sRNA-seq


title: Sample 10_floralBud_nrpd1a-3_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from floralBud_nrpd1a-3 from replicate 3
polIV_fb3.sRNA-seq

value: floral bud


title: Sample 11_globular_dcl234_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from globular_dcl234 from replicate 1
d234_g1.sRNA-seq

value: dicer-like234 triple mutant

value: globular embryo

value: 80% of RNA from 50 embryos


title: Sample 13_globular_dcl234_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from globular_dcl234 from replicate 3
d234_g3.sRNA-seq


title: Sample 9_floralBud_nrpd1a-3_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from floralBud_nrpd1a-3 from replicate 2
polIV_fb2.sRNA-seq


title: Sample 5_earlyHeart_nrpd1a-3_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_nrpd1a-3 from replicate 2
nrpda3.eh_2.sRNA-seq

value: Early heart embryo


title: Sample 17_leaf_dcl234_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_dcl234 from replicate 1
d234_leaf1.sRNA-seq


title: Sample 12_globular_dcl234_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from globular_dcl234 from replicate 2
d234_g2.sRNA-seq


title: Sample 7_floralBud_dcl234_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from floralBud_dcl234 from replicate 3
floralBud_dcl234_Rep3.sRNA-seq


title: WK25_3130_CM_CHW_F_16445

city: Duarte

first: Denis

last: OMeally

state: California

address: 1500 E Duarte Rd

department: Arthur Riggs Diabetes & Metabolism Research Institute

country: USA

institute: City of Hope

description:
Stranded Trueseq library prep; Sequencing run Batch I
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz

data_processing:
Basecalls performed using Illumina RTA version 1.18.64
Raw sequencing reads were processed with the nf-core/RNASeq pipeline version 1.0. Briefly, trimmed reads were mapped using STAR to the GRCm38 reference amended the human MYH11 fusion gene sequence.
Each library was subjected to extensive quality control, including estimation of library complexity, gene body coverage, and duplication rates, among other metrics detailed in the nf-core/rnaseq pipeline repository on GitHub.
Reads were counted across genomic features encoded in the Ensembl GRCm38 annotation using Subread featureCounts and merged into a matrix of counts per gene for each sampled timepoint. CM fusion transcript expression was measured by counting reads that spanned the CM fusion boundary.
Note that in this "validation set" batch effects are apparent due to varying library preparations and confounding of sequencing runs with timepoints, as detailed in the sample description.
Genome_build: GRCm38

organism: Mus musculus

source_name: PBMC

extract_protocol:
AllPrep DNA/RNA Mini Kit (Qiagen)
Sequencing libraries were constructed using the TrueSeq protocol.

treatment_protocol: To induce expression of CM, Cbfb+/56M/Mx1-Cre C57BL/6 mice (4-8 weeks old, including both females and males) were injected with 14 mg/kg poly (I:C) (InvivoGen, San Diego CA) every other day for 7 doses. Similarly treated, wild-type, Cbfb+/56M or Mx1-Cre littermates were used as controls. As previously described, induction of CM expression results in expansion of preleukemic hematopoietic stem and progenitor cell (HSPC) populations in the bone marrow and subsequent development of overt leukemia characterized by >20% cKit+ leukmia blasts, increased white blood cell counts, and splenomegaly. Peripheral blood samples were taken via retro-orital venous sinus before induction and monthly thereafter for the duration of the experiment. All mice were maintained in an AAALAC-accredited animal facility, and all experimental procedures were performed in accordance with federal and state government guidelines and established institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee at the Beckman Research Institute of City of Hope.

tag: strain

tag: timepoint

tag: mouseid

tag: treatment

value: CM

value: CHW

tag: Sex

value: F

value: PBMC


title: Sample 31_BentCotyledon_1ng_MethylC-Seq

description:
Single-end 100 base read MethylC-seq data from BentCotyledon_1ng
BentCotyledon_1ng_MethylC-Seq

value: wild type

value: Bisulfite treated


title: Sample 2_earlyHeart_Col0_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_Col0 from replicate 2
Col.eh_2.sRNA-seq


title: Sample 6_earlyHeart_nrpd1a-3_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_nrpd1a-3 from replicate 3
nrpda3.eh_3.sRNA-seq

value: RNA Polymerase IV mutant


title: Sample 3_earlyHeart_Col0_Rep3.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_Col0 from replicate 3
Col.eh_3.sRNA-seq


title: WK28_3131_CM_CHW_F_16448


title: Sample 4_earlyHeart_nrpd1a-3_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_nrpd1a-3 from replicate 1
nrpda3.eh_1.sRNA-seq


title: Sample 1_earlyHeart_Col0_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from earlyHeart_Col0 from replicate 1
Col.eh_1.sRNA-seq


title: Sample 20_leaf_Hon12_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_Hon12 from replicate 1
leaf_Hon12_Rep1.sRNA-seq

value: 3-weeks old leaf


title: WK18_3200_CM_CHW_M_16451

value: M


title: WK24_3130_CM_CHW_F_16444


title: WK10_3200_CM_CHW_M_16449


title: Sample 27_leaf_suvh456_Rep2.sRNA-seq

description:
Single-end 50 base read small RNA seq data from leaf_suvh456 from replicate 2
leaf_suvh456_Rep2.sRNA-seq

value: histone H3K9 methyltransferases SUVH4,5 and 6 triple mutant


title: WK20_3131_CM_CHW_F_16446


title: WK20_3127_Ctrl_CW_M_16440

value: Ctrl

value: CW


title: WK20_3130_CM_CHW_F_16443


title: Sample 8_floralBud_nrpd1a-3_Rep1.sRNA-seq

description:
Single-end 50 base read small RNA seq data from floralBud_nrpd1a-3 from replicate 1
polIV_fb1.sRNA-seq

value: floral bud


title: WK14_3202_Ctrl_WT_M_16453

value: WT


title: WK18_3202_Ctrl_WT_M_16454


title: WK28_3127_Ctrl_CW_M_16442


title: T6_2686_CM_CHW_M_11845

description:
Stranded Trueseq library prep; Sequencing run Batch H
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: WK14_3200_CM_CHW_M_16450


title: WK10_3202_Ctrl_WT_M_16452


title: T5_2718_CM_CHW_M_11557

description:
Stranded Trueseq library prep; Sequencing run Batch G
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T6_2705_Ctrl_HW_M_11850

value: HW


title: T6_2709_CM_CHW_F_11851


title: T6_2720_Ctrl_HW_M_11852


title: T6_2700_CM_CHW_F_11848


title: WK24_3127_Ctrl_CW_M_16441


title: T6_2702_Ctrl_CW_F_11849


title: T6_2692_Ctrl_CW_F_11847


title: T6_2689_Ctrl_CW_F_11846


title: WK24_3131_CM_CHW_F_16447


title: T6_2685_CM_CHW_M_11844


title: T5_2720_Ctrl_HW_M_11558


title: T5_2705_Ctrl_HW_M_11556


title: T5_2692_Ctrl_CW_F_11552


title: T4_2718_CM_CHW_M_11229

description:
Stranded Trueseq library prep; Sequencing run Batch F
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T4_2720_Ctrl_HW_M_11230


title: T5_2709_CM_CHW_F_11555


title: T4_2705_Ctrl_HW_M_11227


title: T5_2700_CM_CHW_F_11553


title: T5_2702_Ctrl_CW_F_11554


title: T5_2686_CM_CHW_M_11550


title: T5_2685_CM_CHW_M_11549


title: T4_2686_CM_CHW_M_11222


title: T4_2684_CM_CHW_M_11220


title: T4_2702_Ctrl_CW_F_11226


title: T5_2689_Ctrl_CW_F_11551


title: T5_2683_Ctrl_WT_M_11547


title: T3_2719_CM_CHW_M_10895

description:
Stranded Trueseq library prep; Sequencing run Batch E
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T4_2683_Ctrl_WT_M_11219


title: T3_2720_Ctrl_HW_M_10896


title: T4_2700_CM_CHW_F_11225


title: T4_2692_Ctrl_CW_F_11224


title: T3_2718_CM_CHW_M_10894


title: T3_2708_CM_CHW_F_10892


title: T3_2709_CM_CHW_F_10893


title: T3_2705_Ctrl_HW_M_10891


title: T3_2702_Ctrl_CW_F_10890


title: T6_2683_Ctrl_WT_M_11843


title: T3_2692_Ctrl_CW_F_10888


title: T4_2689_Ctrl_CW_F_11223


title: T3_2700_CM_CHW_F_10889


title: T3_2689_Ctrl_CW_F_10886


title: T3_2686_CM_CHW_M_10885


title: T3_2685_CM_CHW_M_10884


title: T3_2683_Ctrl_WT_M_10882


title: T2_2720_Ctrl_HW_M_10822

description:
Stranded Trueseq library prep; Sequencing run Batch D
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T2_2719_CM_CHW_M_10821


title: T2_2731_CM_CHW_F_10823


title: T2_2718_CM_CHW_M_10820


title: T2_2709_CM_CHW_F_10819


title: T2_2705_Ctrl_HW_M_10817


title: T2_2689_Ctrl_CW_F_10812


title: T2_2692_Ctrl_CW_F_10814


title: T2_2690_CM_CHW_F_10813


title: T2_2700_CM_CHW_F_10815


title: T2_2686_CM_CHW_M_10811


title: T2_2685_CM_CHW_M_10810


title: T3_2690_CM_CHW_F_10887


title: T2_2683_Ctrl_WT_M_10808


title: T1_2731_CM_CHW_F_10053

description:
Unstranded TrueSeq library prep; Sequencing run Batch C
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T2_2684_CM_CHW_M_10809


title: T4_2685_CM_CHW_M_11221


title: ZL34_nRBC_494

city: Bethesda

first: Cynthia

middle: E

last: Dunbar

state: Maryland

address: 10 Center Drive

department: Translational Stem Cell Biology Branch

institute: National Heart Lung Blood Institute

data_processing:
Library strategy: targeted sequencing of genomic DNA
Extraction and processing of genetic barcode sequences using custom python code: https://github.com/d93espinoza/barcode_extracter
Genome_build: N/A
Supplementary_files_format_and_content: Processed data matrix in supplementary file contains counts for each genetic barcode (row) for each sample (column).

organism: Macaca mulatta

source_name: Peripheral Blood

extract_protocol:
DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN). 200 ng was used for barcode retrieval PCR with Phusion High-Fidel- ity DNA Polymerase (Thermo Fisher Scientific) via 28 cycles of 98_x0014_C for 10 s, 70_x0014_C for 30 s, 72_x0014_C for 30 s, and then 72_x0014_C for 10 min.
Indexed or non-indexed forward primers and indexed reverse primers were annealed to the amplified DNA containing the barcode sequence.

treatment_protocol: Autologous Transplantation of Lentivirally Barcoded HSPCs: CD34+ HSPCs were collected from ZL34, a 4-year-old male RM, following mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD1300, transduced (MOI = 25) with a barcoded lenti- virus library, and reinfused into the macaque following TBI as described in a protocol approved by the National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee, following all applicable animal care regulations.15–18 ZL34 received weekly dosing with 5 mg/kg cidofovir to suppress CMV reactivation.

tag: cell type

value: nRBC


title: T1_2720_Ctrl_HW_M_10057


title: ZL34_CD34p_553


title: ZL34_nRBC_553


title: ZL34_nRBC_393


title: ZL34_EOS_494

value: EOS


title: T1_2719_CM_CHW_M_10056

value: CM


title: T2_2708_CM_CHW_F_10818

value: CHW


title: T3_2684_CM_CHW_M_10883


title: ZL34_nRBC_266


title: T2_2702_Ctrl_CW_F_10816

value: F


title: T1_2718_CM_CHW_M_10055

value: PBMC


title: ZL34_Mono_494

value: Mono


title: T1_2709_CM_CHW_F_10052


title: ZL34_Mono_553


title: ZL34_Mono_365


title: ZL34_Mono_266


title: T1_2702_Ctrl_CW_F_10050


title: T1_2700_CM_CHW_F_10049


title: T1_2708_CM_CHW_F_10051


title: ZL34_Mono_119


title: ZL34_Mono_187


title: ZL34_nRBC_309


title: ZL34_Mono_32


title: T1_2692_Ctrl_CW_F_10048


title: ZL34_Gr_553

value: Gr


title: ZL34_Gr_494


title: T1_2689_Ctrl_CW_F_10046


title: T1_2690_CM_CHW_F_10047


title: T1_2685_CM_CHW_M_10044


title: T1_2686_CM_CHW_M_10045


title: ZL34_Gr_266


title: ZL34_Gr_365


title: ZL34_Gr_32


title: ZL34_Gr_119


title: T1_2705_Ctrl_HW_M_10054


title: ZL34_B_494

value: B


title: ZL34_Gr_187


title: T0_2719_CM_CHW_M_9775

description:
Unstranded TrueSeq library prep; Sequencing run Batch B
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz


title: T0_2720_Ctrl_HW_M_9776


title: ZL34_B_553


title: ZL34_B_365


title: T0_2731_CM_CHW_F_9772


title: T1_2684_CM_CHW_M_10043


title: ZL34_B_266


title: T0_2709_CM_CHW_F_9771


title: T0_2708_CM_CHW_F_9770


title: ZL34_B_187


title: ZL34_B_393


title: T1_2683_Ctrl_WT_M_10042


title: ZL34_B_119


title: T0_2702_Ctrl_CW_F_9769


title: T0_2700_CM_CHW_F_9768


title: ZL34_B_32


title: ZL34_T_553

value: T


title: ZL34_T_536


title: T0_2689_Ctrl_CW_F_9765


title: T0_2692_Ctrl_CW_F_9767


title: ZL34_T_494


title: ZL34_T_266


title: T0_2718_CM_CHW_M_9774

value: M


title: ZL34_T_393


title: T0_2690_CM_CHW_F_9766


title: ZL34_T_119


title: T0_2685_CM_CHW_M_9763


title: ZL34_T_187


title: L_2731_CM_CHW_F_12128

description:
Unstranded TrueSeq library prep; Sequencing run Batch A
validation cohort
validation_merged_gene_counts__log2cpm_5CPMin2samples.tsv.gz

value: L


title: T0_2684_CM_CHW_M_9762


title: ZL34_T_365


title: T0_2705_Ctrl_HW_M_9773

value: Ctrl


title: T0_2686_CM_CHW_M_9764


title: L_2719_CM_CHW_M_12127


title: ZL34_T_32


title: cip_3h_2

city: Braunschweig

first: Matthias

last: Preusse

address: Inhoffenstr. 7

country: Germany

institute: Helmholtz Centre for Infection Research

data_processing: RNA-seq reads were mapped to the Pseudomonas aeruginosa UCBPP-PA14 (NC_008463.1) genome using the Stampy aligner with default settings.

organism: Pseudomonas aeruginosa

source_name: planktonic culture

growth_protocol: P. aeruginosa PA14 was pre-cultured in in 4 mL lysogeny broth (LB) in glass tubes at 37 °C shaking at 180 rpm. For main experiments a 10 mL culture was prepared from one pre-culture in LB starting at optical density (OD)600 = 0.02.

extract_protocol:
RNA protect buffer (Qiagen) was added to the cells, cells were harvested and RNA was isolated with the RNeasy plus kit (Quiagen). Then mRNA was enriched (MICROBExpress kit (Ambion)), fragmented and ligated to RNA-adapters with a hexameric barcode which enables multiplexing.
After reverse transcription and amplification cDNA was sequenced on an Illumina Genome Analyzer II-x.

treatment_protocol: For ciprofloxacin treatment main cultures were grown until OD600 = 0.5 and then treated for 3 h with sub-inhibitory (sub-MIC) concentrations of ciprofloxacin.

value: Sub-MIC ciprofloxacin


title: wt_3h_2

value: control


title: cip_3h_1


title: T0_2683_Ctrl_WT_M_9761

value: WT


title: L_2690_CM_CHW_F_12124


title: L_2708_CM_CHW_F_12125


title: L_2684_CM_CHW_M_11548


title: HIO donor 2; static/matrigel, T-

city: Mechelen

first: Auste

last: Kanapeckaite

address: General De Wittelaan L11 A3

country: Belgium

institute: Galapagos NV

description:
Donor: Male, 72 years old
8

data_processing:
Trimmomatic 0.36; FASTQ-Processing, Quality-Control; performs a variety of useful trimming tasks
FastQC; sbg:draft-2; analyzes sequence data from FASTQ, BAM, or SAM files. It produces a set of metrics and charts that help identify technical problems with the data.
STAR 2.5.4b; ultrafast universal RNA-seq aligner
Genome_build: Homo_sapiens.GRCh38.98
Supplementary_files_format_and_content: tsv format; raw gene counts

source_name: Human intestinal organoids

growth_protocol: Static: Intestinal crypts were isolated from intestinal biopsies as previously described (Sato et al., 2009, 2011), resuspended in Matrigel (Corning) and polymerized at 37°C. Human intestinal organoids (HIO) were grown in Human intestinal stem cell (HISC) medium (Sato et al., 2009, 2011) containing 50% of Wnt3a conditioned medium and were passaged at a ratio of 1:10 to 1:12 every 10-14 days. Microfluidic: Detailed extracellular matrix (ECM) preparation was performed as previously described (Trietsch et al., 2017). Coated plates were placed at 37°C + 5% CO2 for 15 minutes until the ECM solidified. During this time, HIO were retrieved from Matrigel droplets and made single cells using TryplExpress (Gibco) and mechanical dissociation as previously reported (Sato et al., 2009, 2011). 100 000 HIO cells were seeded in a 2µl volume in the top channel of the OrganoPlate and left to adhere against the gel for 45 min. After incubation, 50 µl of HISC medium devoid of A83-01 (attachment medium) was added to the top inlet and the plates were returned to the incubator at a 70° angle for 4-6 supplementary hours. After 4-6hrs, 50 µl of attachment medium was added to all remaining inlets and outlets and the plates were placed back in the incubator without medium perfusion for 24hrs. On Day 1, plates were placed on an interval rocker (Perfusion Rocker Mini, Mimetas) switching between a +7° and −7° inclination every 8 min (37°C, 5% CO2). Attachment medium was replaced with HISC medium two days after seeding, and HISC medium was refreshed every three days after that.

extract_protocol:
TRI-reagent following manufacturer's instructions.
NEBnext Ultra II RNA Library Prep Kit

treatment_protocol: LPS 100ng/mL, IFN-γ 20ng/mL (T+) for 24hrs

value: colon mucosa

tag: culture

value: static/matrigel

tag: inflammatory trigger

value: None (T-)


title: HIO donor 1; microfluidic, T-

description:
Donor: Male, 50 years old
12

value: rectum mucosa

value: microfluidic


title: HIO donor 1; static/matrigel, T-

description:
Donor: Male, 50 years old
7


title: L_2718_CM_CHW_M_12126

value: CM

value: CHW


title: teNK 4

city: Baltimore

first: Hildur

last: Knutsdottir

address: 733 N Broadway

department: Biomedical Engineering

institute: Johns Hopkins University

description:
This sample includes raw data technical replicate, i.e., same library run in separate flowcell lane. Each technical replicate is represented by a unique SRR.
This sample includes raw data technical replicate, i.e., same library run in separate flowcell lane. Each technical replicate is represented by a unique SRR.
This sample includes raw data technical replicate, i.e., same library run in separate flowcell lane. Each technical replicate is represented by a unique SRR.

data_processing:
Alignment using Hisat2 v 2.1.0, trimming the first base(using option -5 1)
Unique molecular identifiers, UMIs, were marked using NuDup v 2.3
Bam files sorted and filtered based on read quality using samtools
Mapped reads for each gene obtained using HTSeq
Genome_build: grcm38.txt.95
Supplementary_files_format_and_content: Read counts in a text, tab delimited, format

source_name: spleenic NK cells

growth_protocol: For each replicate cells were isolated and cultured in cytokine-supplemented medium for 12–16 h.

extract_protocol:
The cells were snap-frozen, and total RNA was extracted using RNeasy (74104; Qiagen)
The sequencing library was prepared using the TruSeq Stranded mRNA Sample Kit (RS-122- 2101; Illumina)

tag: mouse model

value: MMTV-PyMT

tag: facs

value: spleen

value: NK


title: teNK 3

description: This sample includes raw data technical replicate.


title: teNK 1

description: This sample includes raw data technical replicate, i.e., same library run in separate flowcell lane. Each technical replicate is represented by a unique SRR.


title: teNK 2


title: wt_3h_1


title: hNK 3

value: WT FVB/n


title: HIO donor 2; microfluidic, T-

description:
Donor: Male, 72 years old
3


title: HIO donor 3; microfluidic; T-

description:
Donor: Female, 9 years old
1

value: sigmoid colon mucosa


title: hNK 4


title: HIO donor 3; microfluidic; T+

description:
Donor: Female, 9 years old
2

value: LPS 100ng/mL, IFN-γ 20ng/mL (T+)


title: Sst_adult

city: Dallas

first: Hume

last: Stroud

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern

data_processing:
Fastq files were generated using mkfastq in Cell Ranger (10X Genomics), which generated three fastq files per sample: *I1*=sample index reads; *R1*=10X barcode read and random hexamer (UMI) read; *R2*=transcript read. Biological replicates were aggregated using the Cell Ranger “aggr” pipeline, and mapped to genes using Cell Ranger “count” pipeline (mm10-1.2.0_premrna). The data were clustered using Seurat (Satija et al., 2015) as previously described (Stroud et al., 2017), except that nuclei with at least 500 genes covered were retained for analyses and genes expressed in at least 3 nuclei were kept for analyses. T-distributed stochastic neighbor embedding (t-SNE) plots and gene expression overlays were generated using the “TSNEplot” and “FeaturePlot” functions, respectively. The transcript counts were normalized such that the sum of all genes in a given nuclei equals 10,000.
Genome_build: mm10
Supplementary_files_format_and_content: Sst_KirMUTvsKirWT_sorted.txt.gz  :  Column1= Gene_ID, Column2=Kir2.1MUT reads, Column3 = Kir2.1 WT reads
Supplementary_files_format_and_content: *young_vs_adult*.txt.gz  :   Column1= Gene_ID, Column2=one week reads, Column3 = adult reads

molecule: nuclear RNA

growth_protocol: Brain tissue was taken from wild-type mice or transgenic mice maintained under standard housing conditions.

extract_protocol:
Sst-cre; Sun1 or Vip-cre; Sun1 mouse cortices were harvested and fixed in RNAlater overnight at 4°C. Cortices were homogenized with a dounce-homogenizer and tight pestle 15 times in 5 mL Buffer HB (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH7.8, 1mM DTT, 0.15 mM spermine, 0.5 mM spermidine, Roche protease inhibitor tablets, 10mM Sodium Butyrate, 1 mM Rnase Inhibitor when doing downstream RNA-seq). IGEPAL CA-630 (Sigma) was added to a final concentration of 0.3% to the homogenate, followed by douncing five more times with tight pestle. Homogenized cortices were filtered through a 40 µm strainer, and centrifuged at 500g to pellet the nuclei. The nuclei were resuspended in FACS buffer (1XPBS, 1% BSA) and sorted on GFP-positive nuclei (using Sun1-GFP localizes throughout the nuclear membrane) were sorted into cold FACS buffer using a Sony SH800Z Cell sorter.
All single nuclei RNA sequencing experiments were performed using the Chromium TM Single Cell 3’ Library kit v2 (10X Genomics) following manufacturer instructions.

tag: background

tag: genotype

value: Sst-cre; Sun1

value: Cortex


title: Vip_adult

value: Vip-cre; Sun1

tag: tissue


title: Hela AdMTase-seq (1.0 U Hia5)

city: SEATTLE

first: John

middle: A

last: Stamatoyannopoulos

state: WA

address: 2211 Elliott Avenue, 6th Floor

department: Genome Sciences

institute: Altius Institute / University of Washington

data_processing:
Library strategy: AdMTase-seq
Basecalling, RTA v1.13
Alignments to GRCh38, BWA v0.7.12
Read processing, Samtools v1.2
Read density, Bedops v2.4.15
Peak calling, Hotspot2 v2.1.1
Genome_build: hg38
Supplementary_files_format_and_content: 5% FDR peak calls as .bed file

source_name: Hela cells

growth_protocol: S2-DSRC cells were grown at room temerature to 90% confluency. K562 cells were grown at 37C to 5x10^5 cells/mL; Hela cells were grown at room temerature to 90% confluency

extract_protocol: Short DNA fragments were subjected to standard Illumina library construction

treatment_protocol: Nuclei were extracted using 0.05% IGEPAL in Buffer A (50 mM Tris-HCl, pH 8; 100mM NaCl; 1mM DTT) for 10 minutes at 4 degrees. Nuclei were treated with m6A-Mtase as above.

value: Hela cells

tag: m6a-mtase conditions

value: 1.0 U Hia5 for 20min at 37C

value: Millipore Sigma ABE572


title: Sst_1week


title: K562 AdMTase-seq (0.5 U Hia5)

source_name: K562 cells

value: K562 cells

value: 0.5 U Hia5 for 20min at 37C

tag: antibody


title: K562 AdMTase-seq (1.0 U Hia5)

tag: cell line


title: Sst_Kir2.1WT_rep2

tag: virus

value: Kir2.1 WT AAV


title: HIO donor 3; static/matrigel, T-

description:
Donor: Female, 9 years old
9

value: static/matrigel

value: None (T-)


title: HIO donor 1; microfluidic, T+

description:
Donor: Male, 50 years old
13

value: rectum mucosa

value: microfluidic


title: Hela AdMTase-seq (0.5 U Hia5)


title: Vip_1week


title: HIO donor 2; microfluidic, T+

description:
Donor: Male, 72 years old
4

value: colon mucosa


title: LS-ORGANOID_17-H_P017

city: Houston

first: Eduardo

last: Vilar

state: Texas

address: 1155 Pressler Street

department: Department of Clinical Cancer Prevention

institute: The University of Texas MD Anderson Cancer Center

description: organoid derived from normal colorectal mucosa of patient with Lynch Syndrome (LS)

data_processing:
Sequencing reads quality check and low-quality reads filtering and trimming: patient samples - fastp; patient-derived organoid samples - FastQC; mouse model samples - FastQC.
Alignment: patient samples - STAR_2.4.0e; patient-derived organoid samples - Bowtie; mouse model samples - STAR 2.5.2b
Expression quantification: patient samples - htseq-count; patient-derived organoid samples - rsem-calculate-expression in RSEM v1.2.212; mouse model samples - rsem-calculate-expression in RSEM v1.2.312
Genome_build: patient samples - Ensembl GRCh38;  patient-derived organoid samples - UCSC hg19; mouse model samples - NCBI GRCm38
Supplementary_files_format_and_content: tab-delimited output with raw read counts

source_name: normal colorectal mucosa derived organoid

growth_protocol: For patient-derived organoids, all lines were treated with either DMSO, 500 uM, and 1 mM Naproxen for 48 hours in 24 well plates. For mouse models, treatment and control were administered by oral gavage over a period of 24 weeks; animals were weighted every two weeks and a weight loss >= 20% was an indication for early euthanasia.

extract_protocol:
For patient samples, total RNA was extracted using TriZol (Invitrogen) and PureLink RNA Mini Kit (Invitrogen). RNA was eluted in nuclease free water and concentration was measured using NanoDrop. For patient-derived organoid samples,  total RNA was extracted using TriZol (Invitrogen)  and RNA isolation kit (Ambion. Life Technologies). For mouse samples, RNA was extracted using Direct-zol RNA MiniPrep Plus kit (Zymo Research). RNA was eluted in nuclease free water and concentration was measured using NanoDrop.
Sample preparation, library construction, and sequencing was performed at the Genomics Core at Weill Cornell for patient samples, and at MDACC Sequencing and MicroArray Facility (SMF) for patient-derived organoid and mouse samples.

treatment_protocol: For patient-derived organoid samples, at the end of the treatment and before RNA extraction, organoids were micrographed using brightfield microscope; culture medium was carefully removed and organoids were collected by pipetting several times in cell recovery solution and incubated for 45 min on ice to remove Matrigel by de-polymerization; treated organoids were centrifuged at 300 g for 5 min and pellet is washed once with 10 ml ice cold basal culture medium (DMEM:F12). For mouse models, mice were euthanized, the gut was dissected, flushed with phosphate buffered saline, and stored in RNA-Later at -80C.

tag: group

value: patient-derived organoid

tag: condition

value: naproxen


title: hNK 2

value: WT FVB/n

value: spleen

value: NK


title: LS-ORGANOID_11-H_P011

value: normal colorectal mucosa derived organoid


title: LS-ORGANOID_14-H_P014


title: LS-ORGANOID_2-H_P002


title: LS-ORGANOID_20-H_P020


title: LS-ORGANOID_16-H_P016

value: control


title: LS-ORGANOID_10-H_P010


title: Adult_CTX_VIPcre_Sun1IP_H3K27ac_HiChIP_rep2

data_processing:
library strategy: HiChIP
Paired-end reads were mapped to the mm9 genome using HiC-Pro (Servant et al., 2015). Duplicated reads were removed and assigned to MboI restriction fragments.  Using the HiC-Pro output files, DNA loops were called using Hichipper (Lareau and Aryee, 2018). For each sample, DNA loops called by ten paired-end tags (PET) and FDR<1e-10 were retained. All loops greater than 1 Mb in length were removed as previously performed (Mumbach et al., 2017). Of these high confidence DNA loops, only those that were identified in two independent biological replicates were retained for analyses.
Genome_build: mm9
Supplementary_files_format_and_content: Column 1= region1_chromosome; Column 2= region1_start_coordinate; Column 3= region1_stop_coordinate; Column 4= region2_chromosome; Column 5= region2_start_coordinate; Column 6= region2_stop_coordinate.

extract_protocol:
INTACT-purified nuclei were crosslinked in 1% formaldehyde, followed by the addition of 0.125M glycine to stop crosslinking activity. The nuclei were washed with cold 1XPBS, then lysed once with L1 buffer (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, Vip or Sst-expressing nuclei were INTACT-isolated and cross-linked using 1% formaldehyde as described above. HiChIP experiments were performed as previously described (Mumbach et al., 2016) with the following modifications to the protocol: (1) 4 µl of MboI was used to digest chromatin. (2) Sonication step was performed using Covaris M220 (Millitube) PIP 75; Duty factor 5%; CPB 200; Time 2 minutes. (3) For the IP step, 2 µl of anti-H3K27ac antibodies (Abcam ab4729) and 20 µl of Protein A Dynabeads (Thermofisher) were used.
For adding Illumina sequencing adapters to samples, we used 0.05 µl of Tn5 (Illumina) to each sample and amplified for 12 cycles using the Nextera DNA sample kit (Illumina).

value: Vip-cre; Sun1

value: Cortex

tag: antibodies

value: Rabbit anti-H3K27ac, Abcam, Cat# ab4729


title: Sst_Kir2.1MUT_rep1

value: Sst-cre; Sun1

value: Kir2.1 MUT AAV


title: LS-ORGANOID_5-H_P005


title: Sst_Kir2.1MUT_rep2


title: Adult_CTX_VIPcre_Sun1IP_H3K27ac_HiChIP_rep1


title: hNK 1


title: LS-ORGANOID_13-H_P013


title: 8wk_CTX_VIPcre_Sun1IP_RNA_rep2

data_processing:
Data were analyzed as previously described (Stroud et al., 2017). Briefly, reads were mapped to annotated genes and the genome using Tophat2 (Kim et al., 2013), and quantified with HTseq (Anders et al., 2015). Only uniquely mapping reads were retained, and duplicated reads were discarded.
Genome_build: mm9
Supplementary_files_format_and_content: Gene_ID, read count

extract_protocol:
RNA from INTACT-purified nuclei was extracted with TRIzol (Invitrogen) and purified with a RNeasy kit (on-column DNase treatment) (Qiagen) following manufacturer’s instructions.
Libraries were generated with either the Truseq Strand Specific RNA-Sequencing kit (Illumina) or the NEBNext Ultra Directional RNA Library Prep Kits (NEB).


title: 8wk_CTX_VIPcre_Sun1IP_RNA_rep1


title: Adult_CTX_SSTcre_Sun1IP_H3K27ac_HiChIP_rep2


title: 3wk_CTX_VIPcre_Sun1IP_RNA_rep2


title: 3wk_CTX_VIPcre_Sun1IP_RNA_rep1


title: LS-ORGANOID_4-H_P004


title: LS-ORGANOID_19-H_P019


title: 1wk_CTX_VIPcre_Sun1IP_RNA_rep2


title: 1wk_CTX_VIPcre_Sun1IP_RNA_rep1


title: Sst_Kir2.1WT_rep1

value: Kir2.1 WT AAV


title: LS-PATIENT_2043

description: colorectal normal mucosa from patient with Lynch Syndrome (LS)

source_name: normal colorectal mucosa

value: normal colorectal mucosa

value: patient high-dose naproxen

value: post-treatment


title: LS-PATIENT_1239


title: 8wk_CTX_SSTcre_Sun1IP_RNA_rep2


title: 8wk_CTX_SSTcre_Sun1IP_RNA_rep1


title: LS-PATIENT_8725


title: 3wk_CTX_SSTcre_Sun1IP_RNA_rep2


title: Adult_CTX_SSTcre_Sun1IP_H3K27ac_HiChIP_rep1


title: 3wk_CTX_SSTcre_Sun1IP_RNA_rep1


title: LS-PATIENT_8518


title: LS-PATIENT_9851


title: 1wk_CTX_SSTcre_Sun1IP_RNA_rep2


title: LS-PATIENT_4391


title: 1wk_CTX_SSTcre_Sun1IP_RNA_rep1


title: LS-PATIENT_7858


title: 8wk_CTX_RORBcre_Sun1IP_RNA_rep1

value: Rorb-cre; Sun1


title: LS-PATIENT_9038


title: 8wk_CTX_SSTcre_Sun1IP_BS_rep2

data_processing:
Data was processed as previously described (Stroud et al., 2017). The reads were mapped to the genome using BSmap (Xi and Li, 2009) retaining uniquely mapping, non-redundant reads. Bsmap outputs for CpG sites were calculated.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig
Supplementary_files_format_and_content: text files: column 1= chromosome; column 2=coordinate; column 3 = CG sequence context; column 4= #C calls;  column 5= #T+#C calls

extract_protocol:
Mouse cortices were collected and DNA was extracted using Qiagen Blood and Tissue kit.
WGBS libraries on INTACT-purified nuclei were generated by using a similar method as previously described (Stroud et al., 2017). Briefly, genomic DNA including unmethylated lambda DNA (0.5%; Promega D1521) were sonicated to 200bp using a covaries S2 or M220, and NEXTflex methylated adapters (Bioo Scientific) were ligated onto the sonicated DNA. The libraries were bisulfite-treated using the EZ DNA methylation-Gold kit (Zymo). The final libraries were sequenced on a Nextseq 500.

tag: neuronal subtype

value: Sst neurons


title: 1wk_CTX_RORBcre_Sun1IP_RNA_rep2


title: 1wk_CTX_RORBcre_Sun1IP_RNA_rep1


title: 8wk_CTX_SSTcre_Sun1IP_BS_rep1


title: LS-PATIENT_1778


title: LS-PATIENT_3578


title: LS-PATIENT_1301


title: LS-PATIENT_6093


title: LS-PATIENT_14


title: LS-PATIENT_4737


title: LS-PATIENT_8134


title: 3wk_CTX_VIPcre_Sun1IP_BS

value: Vip neurons


title: P13_CTX_VIPcre_Sun1IP_BS_rep1


title: LS-PATIENT_7586

value: pre-treatment


title: LS-PATIENT_9488


title: P13_CTX_VIPcre_Sun1IP_BS_rep2


title: LS-PATIENT_4575


title: 8wk_CTX_RORBcre_Sun1IP_RNA_rep2


title: P10_CTX_SSTcre_Sun1IP_BS


title: P13_CTX_SSTcre_Sun1IP_BS_rep1


title: LS-PATIENT_8460


title: P10_CTX_VIPcre_Sun1IP_BS_rep1


title: LS-PATIENT_8231


title: LS-PATIENT_6555


title: LS-PATIENT_7886


title: LS-ORGANOID_1-H_P001

value: patient-derived organoid


title: LS-PATIENT_6018


title: LS-PATIENT_1824


title: LS-PATIENT_987


title: LS-PATIENT_2307


title: 1wk_CTX_VIPcre_Sun1IP_BS_rep1


title: 1wk_CTX_VIPcre_Sun1IP_BS_rep2


title: 1wk_CTX_SSTcre_Sun1IP_BS_rep2


title: 15wk_CTX_SSTcre_Dnmt3aKO_Input_BS

value: Sst-cre; Sun1; Dnmt3a fl/fl

value: Mix (whole cortex)


title: 1wk_CTX_SSTcre_Sun1IP_BS_rep1


title: LS-PATIENT_4392


title: 15wk_CTX_SSTcre_Dnmt3aWT_Input_BS

value: Sst-cre; Sun1; Dnmt3a +/+


title: LS-PATIENT_9104


title: P13_CTX_SSTcre_Sun1IP_BS_rep2


title: 13wk_CTX_VIPcre_Dnmt3aKO_Input_BS

value: Vip-cre; Sun1; Dnmt3a fl/fl


title: 13wk_CTX_VIPcre_Dnmt3aKO_Sun1IP_BS

value: Cortex


title: 13wk_CTX_VIPcre_Dnmt3aWT_Input_BS

value: Vip-cre; Sun1; Dnmt3a +/+


title: P10_CTX_VIPcre_Sun1IP_BS_rep2

value: Vip-cre; Sun1


title: LS-PATIENT_1290

value: patient low-dose naproxen


title: LS-PATIENT_8299


title: LS-PATIENT_1430


title: 15wk_CTX_SSTcre_Dnmt3aKO_Sun1IP_BS


title: LS-PATIENT_7933


title: LS-PATIENT_3831


title: 8wk_CTX_genomicDNA_ATAC

data_processing:
Reads were trimmed with Trimmomatic (Bolger et al., 2014) using SLIDINGWINDOW:5:30. Reads were then mapped to the mm9 genome with default parametters using Bowtie2 (Langmead et al., 2009) only retaining uniquely mapping reads with samtools (Li et al., 2009) using parameters -h -b -F 3844 -q 10. Two pseudoreplicates comprising 50% of reads were generated with macs2 randsample. Peaks were called from macs2 with pooled reads and with both pseudoreplicate samples (Zhang, 2008) using parameters -g mm -p 1e-1 --nomodel --extsize 200.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig

extract_protocol:
INTACT-purified nuclei were resuspended in L1 buffer (50mM Hepes pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 0.25% Triton X-100, 0.5% NP40, 10% Glycerol, protease inhibitors, sodium butyrate), and centrifuged at 2000 rpm at 4°C.  L1 buffer was removed, and INTACT-purified nuclei were incubated with Tn5 in TD buffer (Illumina) for 30 minutes at 37°C. To control for potential biases for Tn5 towards specific sequence compositions, we performed the above assay on naked genomic DNA isolated from cortices.
Adapter-ligated DNA fragments were purified using the Qiagen MinElute kit, and subsequently PCR-amplified with the Nextera DNA sample kit using the following conditions: (1) 5 minutes at 72°C, (2) 1 minute at 98°C, (3) 15 seconds at 98°C, (4) 30 seconds at 63°C, (5) one minute at 72°C, (6) Go to step 3 eleven times, (7) hold at 10°C. All ATAC-seq libraries were sequenced on a Nextseq 500.

value: Wild type

value: Mix


title: LS-PATIENT_1263

value: normal colorectal mucosa


title: 13wk_CTX_VIPcre_Dnmt3aWT_Sun1IP_BS


title: Adult_VsCTX_VIPcre_Input_ATAC_rep2

value: Visual cortex


title: 15wk_CTX_SSTcre_Dnmt3aWT_Sun1IP_BS


title: Adult_VsCTX_VIPcre_Input_ATAC_rep1


title: LS-PATIENT_3451

value: post-treatment


title: Adult_VsCTX_VIPcre_Sun1IP_ATAC_rep1


title: Adult_VsCTX_VIPcre_Sun1IP_ATAC_rep2


title: 8wk_CTX_SSTcre_Input_ATAC

value: Sst-cre; Sun1


title: LS-PATIENT_8672


title: 8wk_CTX_VIPcre_Input_ATAC


title: LS-PATIENT_7171


title: 8wk_CTX_RORBcre_Sun1IP_rep2_ATAC

value: Rorb-cre; Sun1

value: Rorb neurons


title: LS-PATIENT_1881


title: LS-PATIENT_8065


title: LS-PATIENT_9799


title: LS-PATIENT_4730


title: 1wk_CTX_RORBcre_Sun1IP_rep2_ATAC


title: LS-PATIENT_1970


title: LS-PATIENT_8750

value: pre-treatment


title: 1wk_CTX_RORBcre_Sun1IP_rep1_ATAC


title: 8wk_CTX_RORBcre_Sun1IP_rep1_ATAC


title: 8wk_CTX_VIPcre_Sun1IP_rep1_ATAC

value: Vip neurons


title: LS-PATIENT_3090


title: LS-PATIENT_5180


title: LS-PATIENT_7172


title: 8wk_CTX_VIPcre_Sun1IP_rep2_ATAC


title: LS-PATIENT_6510


title: 1wk_CTX_VIPcre_Sun1IP_rep2_ATAC


title: LS-PATIENT_1959


title: 1wk_CTX_VIPcre_Sun1IP_rep1_ATAC


title: 8wk_CTX_SSTcre_Sun1IP_rep2_ATAC

value: Sst neurons


title: LS-PATIENT_2557


title: LS-PATIENT_7292


title: 1wk_CTX_SSTcre_Sun1IP_rep2_ATAC


title: LS-PATIENT_4598


title: LS-PATIENT_6572


title: LS-PATIENT_9191


title: LS-PATIENT_6279


title: Adult_Mecp2KO_CTX_IgG_CNR_rep2

data_processing:
library strategy: CUT&RUN
Reads were trimmed with Trimmomatic (Bolger et al., 2014) using SLIDINGWINDOW:5:30. Paired-end trimmed reads were re-paired with the repair.sh utility from bbmap (Bushnell 2014). Reads were then mapped to the mm9 genome using Bowtie2 (Langmead et al., 2009) with parameters as previously detailed (Skene and Henikoff, 2017) using –local –very-sensitive-local –no-unal –no-mixed –no-discordant –phred33 -I 10 -X 700. Only uniquely mapping reads were retained with samtools (Li et al., 2009) using parameters -h -b -F 3844 -q 10.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig

extract_protocol:
Mouse cortices were collected and  homogenized with a dounce-homogenizer and tight pestle 15 times in 5 mL Buffer HB (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH7.8, 1mM DTT, 0.15 mM spermine, 0.5 mM spermidine, Roche protease inhibitor tablets, 10mM Sodium Butyrate, 1mM RNase Inhibitor when doing downstream RNA-seq). IGEPAL CA-630 (Sigma) was added to a final concentration of 0.3% to the homogenate, followed by douncing 5 more times with tight pestle. Homogenized cortices were filtered through a 40um strainer then combined with Working Solution (5 volume of OptiPrep + 1 volume of Diluent [150 mM KCl., 30 mM MgCl2, 120 mM Tricine-KOH pH 7.8] ) at a 1:1 ratio. The mixture was underlaid with 1.2 mL 30% and 1.2 mL 40% OptiPrep gradient solutions (Working solution and diluted with HB Buffer to 30% or 40%). Following ultracentrifugation, 1 mL of nuclei were collected from the interface of the 30% and 40% Optiprep layers. Draq 5 (Abcam) was used to stain DNA. Using a Sony SH800Z Cell sorter, GFP positive nuclei (using Sun1-GFP on nuclear membrane) were selected and sorted into CUT&RUN wash buffer (1 mL of 1M HEPES pH 7.5, 1.5 mL of 5M NaCl, 1 mL of 10% Tween-20, 0.05g BSA, 25 µl of 1M spermidine, protease inhibitors (Roche), H2O to 50 mL). Nuclei were bound to concanavalin A-coated (ConA) beads then washed once with CUT&RUN binding buffer (400 µl of 1M HEPES pH 7.9, 200 µl of 1M KCl, 20 µl of 1M CaCl2, 20 µl of 1M MnCl2, H2O to 50 mL). ConA-bead-bound nuclei were incubated overnight in cold CUT&RUN antibody buffer (1 mL CUT&RUN Triton-wash buffer, 4 µl of 0.5M EDTA) with either in-house anti-FOS antibodies (Emi Ling and Greenberg M.E., in preparation), anti-ARID1A (Cell signaling #12354) or IgG (Cell Signaling #2729S). After incubation, the ConA-bead-bound nuclei were washed once with CUT&RUN antibody buffer, and protein-A-MNase (Skene and Henikoff, 2017) was added to each sample to a final concentration of 700 ng/mL, followed by an incubation for one hour at 4°C. The ConA-bead-bound nuclei were washed twice with CUT&RUN Triton-wash buffer (1 mL of CUT&RUN wash buffer, 10 µl of 10% Triton X-100), and resuspended in 100 µl Triton wash buffer. To each sample, 3 µl of 0.1M CaCl2 was added to each sample, and incubated on ice for 30 min. The reaction was stopped by adding 100 µl 2x STOP buffer (68 µl of 5M NaCl, 40 µl of 0.5M EDTA, 8 µl of EGTA, 4 µl 10% Triton X-100, 2 µl of 1 pg/ul spike-in DNA, 10 µl of RNase A, H2O 868 µl) and incubating at 37°C for 20 minutes. Two µl of 10% SDS was added to the supernatant and the samples were proteinase K treated for > 2 hours at 65°C. DNA was collected by ethanol precipitation.
Library preparation was performed as previously described (Hainer and Fazzio, 2019), except that Rapid Ligase (Enzymatics) was used for adapter ligation, and AMPure XP beads were used for the cleanup of the final libraries. All CUT&RUN libraries were paired-end sequenced (40 bases x 2) on a Nextseq 500.

value: Mecp2 -/y

value: Rabbit IgG; Cell Signaling, Cat# 2729S


title: 1wk_CTX_SSTcre_Sun1IP_rep1_ATAC

value: Cortex


title: LS-PATIENT_6663


title: Adult_Mecp2KO_CTX_H3K27ac_CNR_rep2

value: Rabbit anti-H3K27ac; Abcam, Cat# ab4729


title: Adult_Mecp2WT_CTX_MECP2_CNR_rep2

value: Mecp2 +/y

value: Rabbit anti-MECP2; Greenberg lab in house


title: LS-PATIENT_5585

value: patient low-dose naproxen


title: LS-PATIENT_6448


title: Adult_Mecp2KO_CTX_MECP2_CNR_rep2


title: Adult_Mecp2WT_CTX_H3K27ac_CNR_rep2


title: LS-PATIENT_9100


title: Adult_Mecp2KO_CTX_MECP2_CNR_rep1


title: LS-PATIENT_5514

value: patient placebo


title: 8wk_CTX_SSTcre_Sun1IP_rep1_ATAC


title: Adult_Mecp2KO_CTX_IgG_CNR_rep1


title: Adult_Mecp2WT_CTX_IgG_CNR_rep2


title: Adult_Mecp2WT_CTX_MECP2_CNR_rep1


title: Adult_Mecp2KO_CTX_H3K27ac_CNR_rep1


title: LS-PATIENT_7456

value: normal colorectal mucosa


title: LS-PATIENT_6851


title: LS-PATIENT_5922


title: Adult_Mecp2WT_CTX_IgG_CNR_rep1


title: Adult_Mecp2WT_CTX_H3K27ac_CNR_rep1


title: Adult_CTX_Dnmt3aKO_MECP2_CNR_rep2

value: Nestin-cre; Dnmt3a fl/fl


title: LS-PATIENT_1672

value: post-treatment


title: Adult_CTX_Dnmt3aKO_IgG_CNR_rep2


title: LS-PATIENT_8519


title: Adult_CTX_Dnmt3aKO_H3K27ac_CNR_rep2


title: LS-PATIENT_4127


title: LS-PATIENT_2852


title: Adult_CTX_Dnmt3aWT_MECP2_CNR_rep2

value: Dnmt3a fl/fl


title: LS-PATIENT_5808


title: Adult_CTX_Dnmt3aWT_H3K27ac_CNR_rep2


title: Adult_CTX_Dnmt3aWT_IgG_CNR_rep2


title: LS-PATIENT_1257


title: Adult_CTX_Dnmt3aKO_MECP2_CNR_rep1


title: LS-PATIENT_5219


title: Adult_CTX_Dnmt3aKO_H3K27ac_CNR_rep1


title: Adult_CTX_Dnmt3aKO_IgG_CNR_rep1


title: LS-PATIENT_4833


title: LS-PATIENT_2054


title: LS-PATIENT_4434


title: LS-PATIENT_3029


title: Adult_CTX_Dnmt3aWT_H3K27ac_CNR_rep1


title: Adult_CTX_Dnmt3aWT_IgG_CNR_rep1


title: Adult_CTX_SSTcre_Sun1IP_ARID1A_CNR_rep2

value: Sst-cre; Sun1

value: Rabbit anti-ARID1A; Cell Signaling, Cat# 12354


title: LS-PATIENT_9891


title: LS-PATIENT_7781


title: Adult_CTX_SSTcre_Sun1IP_FOS_CNR_rep2

value: Rabbit FOS; Greenberg lab in house


title: LS-PATIENT_195


title: LS-PATIENT_5035


title: LS-PATIENT_9403


title: LS-PATIENT_9781

value: pre-treatment


title: 1wk_CTX_SSTcre_Sun1IP_ARID1A_CNR_rep3


title: LS-PATIENT_7094


title: 1wk_CTX_SSTcre_Sun1IP_FOS_CNR_rep3


title: 1wk_CTX_SSTcre_Sun1IP_ARID1A_CNR_rep2


title: 1wk_CTX_SSTcre_Sun1IP_IgG_CNR_rep3


title: 1wk_CTX_SSTcre_Sun1IP_FOS_CNR_rep2


title: LS-PATIENT_6889


title: LS-PATIENT_6149


title: LS-PATIENT_9442


title: 1wk_CTX_SSTcre_Sun1IP_IgG_CNR_rep2


title: LS-PATIENT_512


title: LS-PATIENT_2439


title: Adult_CTX_SSTcre_Sun1IP_ARID1A_CNR_rep1


title: LS-PATIENT_7554


title: Adult_CTX_SSTcre_Sun1IP_FOS_CNR_rep1


title: LS-PATIENT_8324


title: Adult_CTX_SSTcre_Sun1IP_IgG_CNR_rep1


title: Adult_CTX_Dnmt3aWT_MECP2_CNR_rep1


title: LS-PATIENT_3754


title: 1wk_CTX_SSTcre_Sun1IP_ARID1A_CNR_rep1


title: LS-PATIENT_5371


title: Adult_CTX_SSTcre_Sun1IP_IgG_CNR_rep2


title: 1wk_CTX_SSTcre_Sun1IP_FOS_CNR_rep1


title: LS-PATIENT_744


title: LS-PATIENT_3164


title: 1wk_CTX_SSTcre_Sun1IP_IgG_CNR_rep1


title: 3wk_CTX_VIPcre_Sun1IP_FOS_CNR_rep2

value: Vip-cre; Sun1


title: LS-PATIENT_4709


title: 3wk_CTX_VIPcre_Sun1IP_IgG_CNR_rep2


title: LS-PATIENT_8215


title: 3wk_CTX_VIPcre_Sun1IP_FOS_CNR_KA_rep1

tag: treatment

value: Kainic acid treated


title: 3wk_CTX_VIPcre_Sun1IP_FOS_CNR_KA_rep2


title: 3wk_CTX_VIPcre_Sun1IP_FOS_CNR_rep1


title: 3wk_CTX_VIPcre_Sun1IP_IgG_CNR_KA_rep1

value: Rabbit IgG; Cell Signaling, Cat# 2729S


title: LS-PATIENT_2942


title: 3wk_CTX_SSTcre_Sun1IP_ARID1A_CNR

value: Cortex


title: LS-PATIENT_6910


title: LS-PATIENT_2612


title: LS-PATIENT_7152


title: 3wk_CTX_SSTcre_Sun1IP_FOS_CNR_rep2


title: 3wk_CTX_SSTcre_Sun1IP_IgG_CNR_rep2


title: 3wk_CTX_SSTcre_Sun1IP_FOS_CNR_rep1


title: Total 80S Ribosome FPs Hela cells 2 min Harringtonine [6_80S_NIH3T3_T4]

city: Heidelberg

first: Jonathan

last: Bohlen

address: Im Neuenheimer Feld 581

institute: DKFZ

description:
MM_80S_Total_T4
processed data file: Master_Data_Table.xlsx

data_processing:
Library strategy: Ribo-seq
Adapter sequences and randomized nucleotides (Nextflex) or polyA stretches (Clonentech) were trimmed from raw reads using cutadapt.
Nuclear and mitochondrial Ribosomal RNA and tRNA reads were removed by alignment to human tRNA and rRNA sequences using bowtie2.
The remaining reads were separately aligned to the human transcriptome (Ensemble transcript assembly 94) and human genome using BBmap.  Multiple mappings were allowed.
Genome_build: human transcriptome (Ensemble transcript assembly 94)
Supplementary_files_format_and_content: Master_Data_Table1.xlsx: Excel file includes values that are normalized to sequencing depth (divided by number of mappings and multiplied by a scaling factor).

growth_protocol: HeLa cells were cultured in DMEM +10% fetal bovine serum +100 U/ml Penicillin/Streptomycin (Gibco 15140122).

extract_protocol:
For cell harvest, growth medium was poured off and cells were quickly washed with ice-cold  washing solution (1x PBS 10 mM MgCl2 800 µM Cycloheximide). Washing solution was immediately poured off and freshly prepared crosslinking solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 0.025% PFA, 0.5 mM DSP) was added to the cells. Cells were incubated with crosslinking solution for 15 minutes at room temperature while slowly rocking. Crosslinking solution was then poured off and remaining crosslinker was inactivated for 5 minutes with ice-cold quenching solution (1x PBS, 10 mM MgCl2, 200 µM Cycloheximide, 300 mM Glycine). Quenching solution was poured off and 150 µl of lysis buffer (0,25 M HEPES pH 7.5, 50 mM MgCl2, 1 M KCl, 5% NP40, 1000 μM CHX) was added to each 15 cm dish, resulting in 750µL of lysate. Lysis was carried out at 4°C.
After RNA extraction from total and IP-purified fractions, RNA quality and integrity were determined on an Agilent Bioanalyzer using the total RNA Nano 6000 Chip. For size selection, RNA was run on 15% Urea-Polyacrylamide gels (Invitrogen) and fragments of size 20-60 nt (80S libraries) and 20-80 nt (40S libraries) were excised using the Agilent small RNA ladder as a reference. RNA was extracted from the gel pieces by smashing the gels into small pieces with gel smasher tubes and extracting the RNA in 0.5 ml of 10 mM Tris pH 7 at 70°C for 10 minutes. Gel pieces were removed and RNA was precipitated using Isopropanol.  Footprints were then dephosphorylated using T4 PNK (NEB) for 2 hours at 37°C in PNK buffer without ATP. Footprints were then again precipitated and purified using isopropanol. For 40S footprints, contaminating 18S rRNA fragments were depleted as follows. Prevalent 18S rRNA fragments from the first round of 40S footprinting were used to design complementary Biotin-TEG-DNA oligonucleotides (sequences listed in Supplemental Table 4, ordered from Sigma-Aldrich). 100ng of RNA footprints were then hybridized to a mixture (proportional to occurrence of the fragment, listed in Suppl. Table 4) of these DNA oligos (in 40x molar excess) in (0.5M NaCl, 20mM Tris pH7.0, 1mM EDTA, 0.05% Tween20) by denaturing for 90 sec at 95C and then annealing by reducing the temperature by -0.1C/sec down to 37°C, then incubating 15min at 37°C. Hybridized species were pulled out using Streptavidin magnetic beads (NEB) by incubating at room temperature for 15 minutes, and the remaining RNA was purified by isopropanol precipitation. Footprints were then assayed using an Agilent Bioanalyzer small RNA chip and Qubit smRNA kit. 25 ng or less of footprint RNA was used as input for library preparation with SMARTer smRNA-SeqKit for Illumina from Takara / Clontech Laboratories according to the manufacturer’s instructions. Deep-sequencing libraries were sequenced on the Illumina Next-Seq 550 system.

treatment_protocol: Cellular stress conditions were induced by treatment with Tunicamycin at 1 µg / ml or 250 ng / ml for 16 hours.

tag: rrna depletion

tag: library preparation kit

value: Takara

tag: index name

tag: index sequence

value: CTGAAGCT


title: 3wk_CTX_VIPcre_Sun1IP_IgG_CNR_KA_rep2


title: eiF4E selectve 80S Ribosome FPs, Untreated NIH3T3 Cells [6_80S_NIH3T3_eIF4E]

description:
MM_80S_eIF4E
processed data file: Master_Data_Table.xlsx

value: TAATGCGC


title: eiF4E selectve 40S Ribosome FPs, Untreated NIH3T3 Cells [6_40S_NIH3T3_eIF4E]

description:
MM_40S_eIF4E
processed data file: Master_Data_Table.xlsx

value: GAGATTCC


title: LS-PATIENT_3402

value: patient placebo


title: 3wk_CTX_SSTcre_Sun1IP_IgG_CNR_rep1


title: Total 80S Ribosome FPs Hela cells [6_80S_NIH3T3_UT]

description:
MM_80S_Total_T0
processed data file: Master_Data_Table.xlsx

value: ATTCAGAA


title: 3wk_CTX_SSTcre_Sun1IP_Input_HYBRID

data_processing:
Reads were trimmed with Trimmomatic (Bolger et al., 2014) using SLIDINGWINDOW:5:30. Reads were then mapped to the mm9 genome with default parameters using Bowtie2 (Langmead et al., 2009) only retaining uniquely mapping reads with samtools (Li et al., 2009) using parameters -h -b -F 3844 -q 10. For the allele-specific mapping of C57BL6/J and SPRET/EiJ reads, the SPRET/EiJ pseudogenome was assembled as detailed in Vierbuchen et al., 2017. Briefly, SNPs in the SPRET/EiJ relative to the mm10 reference genome were annotated with VCFtools, only high-confidence SNPs were retained, and these SNPs were used to construct the SPRET/EiJ pseudogenome with Modtools. Reads were trimmed with Trimmomatic (Bolger et al., 2014) using SLIDINGWINDOW:5:30. Paired-end trimmed reads were re-paired with the repair.sh utility from bbmap (Bushnell 2014). Each read was mapped in parallel to both the mm10 reference genome or the generated SPRET/EiJ pseudogenome. Reads initially mapped to the SPRET/EiJ pseudogenome were converted back to mm10 reference genome coordinates with Lapels (Huang et al., 2014). BAM files with mapped reads of reads that overlapped at least one SPRET/EiJ SNP were retained for downstream analysis. These reads converted back into FASTQ files wherein every possible allelic combination of SNPs for each read was represented (up to 2^20 reads). The original reads were only retained if and only if all possible allelic combinations mapped to the same coordinates on the alternate genome.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig

extract_protocol:
INTACT-purified nuclei were crosslinked in 1% formaldehyde, followed by the addition of 0.125M glycine to stop crosslinking activity. The nuclei were washed with cold 1XPBS, then lysed once with L1 buffer (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 0.25% Triton X-100, 0.5% NP40, 10% Glycerol, protease inhibitors, sodium butyrate), followed by an incubation in L2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl, protease inhibitors, sodium butyrate). The nuclei were resuspended in L3 buffer (1M Tris-HCl pH8.0, 5M NaCl, 0.5 M EDTA, 0.5 M EGTA, 10% Na-Deoxycholate, 20% N-lauroylsarcosine, protease inhibitors, sodium butyrate) and sonicated with a Bioruptor (Diagenode) for 40 cycles of 30 seconds “on” and 45 seconds “off.” Sonicated chromatin were centrifuged at 13,000 rpm for 10 minutes at 4°C to remove insoluble pellet, and then pre-cleared by incubating with Protein A Dynabeads (Thermofisher) for 2 hours at 4°C. Pre-cleared chromatin were incubated with Dynabeads conjugated to H3K27ac (Abcam ab4729) or H3K4me1 (Abcam ab8895) antibodies overnight at 4°C. Beads were washed twice each with Low Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 150mM NaCl, 2mM EDTA), High Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 500mM NaCl, 2mM EDTA) and LiCl buffer (250mM LiCl, 1% NP40, 1mM EDTA, 10mM TrisHCl pH8.0, 1% Sodium Deoxycholate) at 4°C, and washed once with 1XTE buffer at room temperature. Immunoprecipitated chromatin were first eluted off the beads with 1XTE+1%SDS at 65°C with shaking for 30 minutes, and then were de-crosslinked overnight at 65°C. RNAseA was added to each sample, followed by an incubation at room temperature for 30 minutes. The samples were incubated with proteinase K for two to three hours at 55°C with shaking. Immunoprecipitated DNA were purified using phenol-chloroform-isoamyl alcohol followed by Qiagen MinElute columns.
Libraries were constructed by using Ovation Ultralow Library Systems (Nugen) following manufacturer instructions.

value: C57BL/6 x SPRET/EiJ F1


title: LS-PATIENT_6733

value: normal colorectal mucosa


title: 3wk_CTX_VIPcre_Sun1IP_IgG_CNR_rep1


title: 3wk_CTX_SSTcre_Sun1IP_H3K27ac_HYBRID_rep1

value: Rabbit anti-H3K27ac, Abcam, Cat# ab4729


title: 3wk_CTX_SSTcre_Sun1IP_H3K27ac_HYBRID_rep2

value: Sst-cre; Sun1


title: Total 40S Ribosome FPs NIH3T3 cells 4 min Harringtonine [6_40S_NIH3T3_T4]

description:
MM_40S_Total_T4
processed data file: Master_Data_Table.xlsx

value: CGCTCATT


title: Adult_VsCTX_VIPcre_Sun1IP_H3K27ac_0hr_rep2

value: Visual cortex

value: Dark reared


title: LS-PATIENT_2436

value: pre-treatment


title: Total 40S Ribosome FPs NIH3T3 cells  [6_40S_NIH3T3_UT]

description:
MM_40S_Total_T0
processed data file: Master_Data_Table.xlsx

value: ATTACTCG


title: Adult_VsCTX_VIPcre_Sun1IP_H3K27ac_0hr_rep1

value: Vip-cre; Sun1


title: Adult_VsCTX_VIPcre_Sun1IP_H3K27ac_3hr_rep1

value: Dark reared, then exposed to light for 3 hours


title: Adult_VsCTX_VIPcre_Sun1IP_H3K27ac_3hr_rep2


title: WT rep3 [N393-0M]

city: Stanford

first: Y. Lucy

last: Liu

state: CA

address: 240 Pasteur Drive, BMI Suite 2550

department: Pediatrics

institute: Stanford Medical School

description: N393-0M_TSTRLT_1d7d4cb7_GCL-YL-P0054-SB0110_S2

data_processing:
BCL to FastQ file conversion with bcl2fastq2 v2.18.0.12 (default Illumina adapter trimming performed during conversion).
STAR v2.5.3a was used to align each sample’s paired-end reads to the Ensembl Mus Musculus reference genome build GRCm38 (using STAR’s 2-pass method)
Picard v2.0.1 was used to add read group information. Aligned files were also processed using Sambamba v0.6.5 to mark duplicates and sort reads.
Each sample’s BAM file was initially processed using StringTie v1.3.3b, using Ensembl gene annotations to guide transcriptome (limiting output to only annotated genes). The StringTie option to output “Ballgown-ready” files was enabled.
Using R v3.5.0, Ballgown-ready files were imported into DESeq2 v1.22.2 (using tximport v1.10.1).
Genome_build: Ensembl Mus musculus (GRCm38)
Supplementary_files_format_and_content: tab-delimited text files include raw/unnormalized gene count data for each sample.

source_name: WT

extract_protocol:
RNA-seq samples were prepared using the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina, catalog number 20020599) according to manufacturer recommendations.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: genotype/variation

value: wild type


title: WT rep2 [N412-0M]

description: N412-0M_TSTRLT_21c88b76_GCL-YL-P0054-SB0110_S6


title: WT rep1 [N411-4M]

description: N411-4M_TSTRLT_3bbc53ab_GCL-YL-P0054-SB0110_S5


title: JMML rep3 [N393-2M]

description: N393-2M_TSTRLT_a787eb5f_GCL-YL-P0054-SB0110_S3

source_name: JMML

value: Juvenile myelomonocytic leukemia (JMML)


title: Total 40S Ribosome FPs NIH3T3 cells 2 min Harringtonine [6_40S_NIH3T3_T2]

description:
MM_40S_Total_T2
processed data file: Master_Data_Table.xlsx

value: Takara

value: TCCGGAGA


title: JMML rep2 [N409-0F]

description: N409-0F_TSTRLT_23fdf76f_GCL-YL-P0054-SB0110_S4


title: 3wk_CTX_SSTcre_Sun1IP_H3K27ac_HYBRID_rep3

tag: background

value: Cortex


title: P10_CTX_VIPcre_Sun1IP_H3K4me1

tag: genotype

value: Rabbit anti-H3K4me1, Abcam, Cat # ab8895


title: P10_CTX_VIPcre_Sun1IP_H3K27ac


title: JMML rep1 [N348-0F]

description: N348-0F_TSTRLT_6b1f26b1_GCL-YL-P0054-SB0110_S1


title: P10_CTX_SSTcre_Sun1IP_H3K4me1


title: P10_CTX_SSTcre_Sun1IP_H3K27ac


title: P13_CTX_VIPcre_WT_Sun1IP_H3K4me1


title: RNA-seq from Untreated NIH3T3 cells [6_RNA-seq_NIH3T3]

description:
MM_RNA_seq
processed data file: Master_Data_Table.xlsx

data_processing:
Adapter sequences and randomized nucleotides (Nextflex) or polyA stretches (Clonentech) were trimmed from raw reads using cutadapt.
Nuclear and mitochondrial Ribosomal RNA and tRNA reads were removed by alignment to human tRNA and rRNA sequences using bowtie2.
The remaining reads were separately aligned to the human transcriptome (Ensemble transcript assembly 94) and human genome using BBmap.  Multiple mappings were allowed.
Genome_build: human transcriptome (Ensemble transcript assembly 94)
Supplementary_files_format_and_content: Master_Data_Table1.xlsx: Excel file includes values that are normalized to sequencing depth (divided by number of mappings and multiplied by a scaling factor).

value: Ribo Zero Gold

value: GAGATTCC


title: yeast eIF3a bound 80S 3-1

data_processing:
Library strategy: SelTCP-seq
Illumina CASAVA 1.8 for base calling
adapter trimming (fastx_clipper of FASTX Toolkit 0.0.13), filtering by alignment to rRNA (bowtie 1.2.2) and ncRNA, tRNA (bowtie2 2.2.6)
alignment to transcriptome Ensembl R64-1-1.80 (with 3’ UTR, 5’ UTR, and 5’ UTR intron annotation from SGD) and genome R64-1-1 with TopHat 2.1.0
Genome_build: R64-1-1
Supplementary_files_format_and_content: metagene_FP5end_AUG_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the AUG start codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_FP5end_STOP_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the STOP codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_coverage_CAP_-40+40.csv; footprint coverage from -40 nt to +40 nt relative to the transcript start
Supplementary_files_format_and_content: genewise_FPcount_transcript_area.csv; footprint count per transcript area
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints without 5’extension (footprint 5’end from -12 to -14 nt)
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50_all_AUG_associated_FPs.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints
Supplementary_files_format_and_content: coverage_GCN4_YEL009C_-50.csv; coverage for the 5’UTR and the beginning of the CDS for GCN4 (starting at -50 nt from the transcript start)
Supplementary_files_format_and_content: coverage_for_co-translational_assembly.ods; coverage for eIF3a, eIF3b, eIF3g and eIF2beta of 80S footprints and eIF3a/c bound 80S footprints (starting at -50 nt from transcript start)

organism: Saccharomyces cerevisiae

source_name: cell culture

growth_protocol: in 1 litre YPD at 30ºC up to OD600 of ~0.7-0.8

extract_protocol:
cell lysis with bead beater, RNase I digestion to generate ribosomal footprints, 40S/80S separation (followed by Co-IP if indicated), hot acid phenol RNA extraction
library construction according to Ingolia et al., 2010, Methods Enzymol (different RNA fragment size selection, different set of rRNA depletion oligos)

treatment_protocol: incubation of the cell culture with 0.8 % w/v formaldehyde on ice for 1 h

tag: strain

value: SY182 (eIF3a/TIF32-FLAG)

tag: rna fragment size selection

value: approximately 18 to 85

tag: co-ip

value: yes (anti-FLAG, Sigma A2220)

tag: protocol

value: RNA 80S footprint


title: yeast eIF3a bound 80S 1-1


title: yeast eIF3a bound 80S 2-1


title: Schwann-like cells_0hr_Control_rep2

type: RNA

city: Birmingham

first: Zsuzsanna

last: Nagy

address: Medical School

country: United Kingdom

institute: University of Birmingham

description: Gene expression after 24 hr in Culture (High Glucose DMEM+10%FCS), human Schwann-like cells

hyb_protocol: Cy3-labelled cRNA was fragmented at following the manufacturers instructions. On completion of the fragmentation reaction, the Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent G4851C SurePrint G3 Human Gene Expression v3 Microarray. After hybridization, the arrays were washed then dried immediately.

scan_protocol: Slides were scanned immediately after washing on the Agilent Technologies Scanner G2505C using one colour scan setting.

data_processing: The scanned images were analysed with the Feature Extraction Software of Agilent using default parameters (protocol GE1_107_Sep09 and Grid: 072363_D_F_20150612) to obtain background subtracted and spatially detrended Processed Signal intensities.   The background corrected signal was log2 transformed. To reduce the false discovery rate in the analysis, probes where the log2 signal was below 5 were removed. Additionally duplicate probes were removed before statistical analysis.

source_name: Schwann-like cells, 0hr, Control culture, replicate 2

label_protocol: Cyanine-3 (Cy3) labelled cRNA was prepared sing the One-Color Low Input Quick Amp Labelling Protocol One-Color Low RNA Input kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: Eight 25 cm2 flasks were coated by adding 2 ml per flask of a solution of 50µg/ml poly-d-lysine in HBSS and incubating overnight at 37°C in the dark. Flasks were washed with cell culture water and air-dried for 30 min in the dark. Flasks were coated by adding 1 ml per flask of a solution of 25µg/ml laminin in PBS and incubating for 2 hour at 37°C in the dark. The laminin flasks were washed with PBS then three times before plating cells. Schwann cells growth medium was prepared by adding 10% of FBS to high-glucose DMEM and pre-warmed to 37°C. Cells were thawed in a 37°C water bath for no longer than 2 min.   Cells from 12 vials (ATCC-CRL-2884) were each gently transferred to a tube containing 10 ml of high-glucose DMEM medium and centrifuged at 400 RCF for 10 min. Pellet was resuspended in culture medium. The cells from the 12 vials were mixed and distributed equally (5 ml each flask) in Culture medium (CM made with high-glucose DMEM+10% FCS) into the previously coated 25 cm2 flasks (n=8).   Cells were incubated at 37°C with 5% CO2. Cells were allowed to settle for 24 hours before drug treatment.

extract_protocol: Culture media were removed and cells were harvested in 2.5 ml Trizol:Water (4:1) solution containing 0.5 ml of chloroform at room temperature (RT). The flasks were inspected under the microscope to ensure full removal of cells. The collected lysates were stored at -80 C. Trizol RNA extraction was performed according to manufacturers recommendations. The RNA was precipitated from the aqueous phase with 100% ethanol, washed twice and was eluted in 50 μL warm RNase-Free Water. RNA integrity was assessed on the Agilent Bioanalyzer 2100 using the RNA 6000 Nano Assay. The RIN number of  the samples was above 7.5. 2nd QC was performed using the Nanodrop ND-1000 Spectrometer Analysis. RNA quality and quantity was adequate for microarray analysis.

treatment_protocol: A fresh 100X ILB stock (1.0 mg/ml) was prepared in DMEM-F12. The DMEM-F12 was sterile. The concentrated ILB stock was sterile filtered, and added to the culture media (CM) to make a 2x concentrated ILB solution (19.6 ml CM and 0.4 ml ILB stock). The Control was made using 19.6 ml CM and 0.4 ml of DMEM-F12. Drug and Control media was added to the respective flasks (5 ml each) to reach the 0.01mg/ml concentration of ILB in each dish (with a total of 10 ml CM each). The cultures were incubated for 48 hours at 37 ºC in a humidified incubator with 5% CO2.

tag: cell line

value: Human Schwann-like cells ATCC-CRL-2884

value: 0hr, Control culture


title: Schwann-like cells_48hr_ILB_rep3

description: Gene expression after 72 hr, 24 hours in Control conditions (High Glucose DMEM+10%FCS) and 48 hours treatment condition (High Glucose DMEM+10%FCS+0.01mg/ml ILB), human Schwann-like cells

source_name: Schwann-like cells, 48hr, ILB treated culture , replicate 3

value: 48hr, ILB treated culture


title: Schwann-like cells_48hr_ILB_rep2

source_name: Schwann-like cells, 48hr, ILB treated culture , replicate 2


title: Schwann-like cells_0hr_Control_rep1

source_name: Schwann-like cells, 0hr, Control culture, replicate 1


title: LS-MOUSE_VilarM01

description: small intestinal normal mucosa of Lynch Syndrome (LS) mouse model

source_name: normal intestinal mucosa

tag: tissue

value: normal intestinal mucosa

tag: group

value: LS mouse model

value: naproxen


title: yeast eIF3c bound 80S 1-2

value: H25 (eIF3c/NIP1-FLAG)


title: P13_CTX_VIPcre_WT_Sun1IP_H3K27ac

value: Vip-cre; Sun1

tag: antibodies

value: Rabbit anti-H3K27ac, Abcam, Cat# ab4729


title: Schwann-like cells_48hr_Control_rep1

description: Gene expression after 72 hr in Culture (High Glucose DMEM+10%FCS), human Schwann-like cells

source_name: Schwann-like cells, 48hr, Control culture, replicate 1

value: 48hr, Control culture


title: Schwann-like cells_48hr_Control_rep3

source_name: Schwann-like cells, 48hr, Control culture, replicate 3


title: Schwann-like cells_48hr_ILB_rep1

source_name: Schwann-like cells, 48hr, ILB treated culture , replicate 1


title: yeast unselected 80S 2-1

data_processing:
Library strategy: TCP-seq
Illumina CASAVA 1.8 for base calling
adapter trimming (fastx_clipper of FASTX Toolkit 0.0.13), filtering by alignment to rRNA (bowtie 1.2.2) and ncRNA, tRNA (bowtie2 2.2.6)
alignment to transcriptome Ensembl R64-1-1.80 (with 3’ UTR, 5’ UTR, and 5’ UTR intron annotation from SGD) and genome R64-1-1 with TopHat 2.1.0
Genome_build: R64-1-1
Supplementary_files_format_and_content: metagene_FP5end_AUG_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the AUG start codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_FP5end_STOP_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the STOP codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_coverage_CAP_-40+40.csv; footprint coverage from -40 nt to +40 nt relative to the transcript start
Supplementary_files_format_and_content: genewise_FPcount_transcript_area.csv; footprint count per transcript area
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints without 5’extension (footprint 5’end from -12 to -14 nt)
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50_all_AUG_associated_FPs.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints
Supplementary_files_format_and_content: coverage_GCN4_YEL009C_-50.csv; coverage for the 5’UTR and the beginning of the CDS for GCN4 (starting at -50 nt from the transcript start)
Supplementary_files_format_and_content: coverage_for_co-translational_assembly.ods; coverage for eIF3a, eIF3b, eIF3g and eIF2beta of 80S footprints and eIF3a/c bound 80S footprints (starting at -50 nt from transcript start)

value: no


title: yeast mRNA 1-1

data_processing:
Illumina CASAVA 1.8 for base calling
adapter trimming (fastx_clipper of FASTX Toolkit 0.0.13), filtering by alignment to rRNA (bowtie 1.2.2) and ncRNA, tRNA (bowtie2 2.2.6)
alignment to transcriptome Ensembl R64-1-1.80 (with 3’ UTR, 5’ UTR, and 5’ UTR intron annotation from SGD) and genome R64-1-1 with TopHat 2.1.0
Genome_build: R64-1-1
Supplementary_files_format_and_content: metagene_FP5end_AUG_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the AUG start codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_FP5end_STOP_-80+30_FPlength.tbl; counts of footprint 5’ ends positioned from -80 nt to +30 nt relative to the STOP codon (over rows) respecting the footprint length (18 – 98 nt over columns)
Supplementary_files_format_and_content: metagene_coverage_CAP_-40+40.csv; footprint coverage from -40 nt to +40 nt relative to the transcript start
Supplementary_files_format_and_content: genewise_FPcount_transcript_area.csv; footprint count per transcript area
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints without 5’extension (footprint 5’end from -12 to -14 nt)
Supplementary_files_format_and_content: genewise_mean_CI_FP5end_FP3end_AUG_-50+50_all_AUG_associated_FPs.ods; distribution (mean and 95 percentiles) of footprint 5’end and 3’end positions relative to the AUG start codon of start codon associated footprints
Supplementary_files_format_and_content: coverage_GCN4_YEL009C_-50.csv; coverage for the 5’UTR and the beginning of the CDS for GCN4 (starting at -50 nt from the transcript start)
Supplementary_files_format_and_content: coverage_for_co-translational_assembly.ods; coverage for eIF3a, eIF3b, eIF3g and eIF2beta of 80S footprints and eIF3a/c bound 80S footprints (starting at -50 nt from transcript start)

extract_protocol:
cell lysis with bead beater, total RNA extraction with RNeasy Mini Kit, poly(A) selection with poly(A)Purist MAG Kit, fragmentation
library construction according to Ingolia et al., 2010, Methods Enzymol (different RNA fragment size selection, different set of rRNA depletion oligos)

value: RNA-seq


title: yeast unselected 80S 3-1


title: P13_CTX_SSTcre_WT_Sun1IP_H3K4me1

value: Sst-cre; Sun1

value: Cortex

value: Rabbit anti-H3K4me1, Abcam, Cat # ab8895


title: LS-MOUSE_VilarM11

tag: condition


title: yeast unselected 80S 1-3

value: SY194 (eIF2beta/SUI3-FLAG)

value: approximately 18 to 85


title: LS-MOUSE_VilarM15


title: Schwann-like cells_48hr_Control_rep2

source_name: Schwann-like cells, 48hr, Control culture, replicate 2

value: Human Schwann-like cells ATCC-CRL-2884

tag: treatment


title: yeast unselected 80S 1-1

value: SY182 (eIF3a/TIF32-FLAG)

value: RNA 80S footprint


title: P13_CTX_SSTcre_WT_Sun1IP_H3K27ac


title: yeast unselected 80S 1-2


title: Adult_CTX_VIPcre_Dnmt3aflfl_Sun1IP_H3K27ac_rep2

value: Vip-cre; Sun1; Dnmt3a fl/fl


title: LS-MOUSE_VilarM03

value: normal intestinal mucosa

value: LS mouse model

value: control


title: yeast unselected 40S 1-3

value: RNA 40S footprint


title: yeast eIF3c bound 40S 1-2

value: H25 (eIF3c/NIP1-FLAG)

value: yes (anti-FLAG, Sigma A2220)


title: yeast eIF3a bound 40S 1-1


title: Adult_CTX_VIPcre_Dnmt3aflfl_Sun1IP_H3K4me1_rep2


title: Adult_CTX_VIPcre_Dnmt3aWT_Sun1IP_H3K27ac_rep2

value: Vip-cre; Sun1; Dnmt3a +/+

value: Rabbit anti-H3K27ac, Abcam, Cat# ab4729


title: LS-MOUSE_VilarM07


title: PR8;PB1-UFO_∆_2

city: La Jolla

first: Max

last: Chang

address: 9500 Gilman Drive

institute: University of California, San Diego

data_processing:
Raw sequencing reads were aligned to the mouse GRCm38 genome with STAR
Read counts per gene were calculated using FeatureCounts
DESeq2 was used to generate FPKM values
Genome_build: GRCm38
Supplementary_files_format_and_content: comma-separated values containing FPKM quantification for all samples

extract_protocol:
Mouse lungs were homogenized in liquid nitrogen. 1 ml Trizol was added per 50-100 mg of tissue and the homogenate was incubated at room temperature for 5 minutes. 200 μl chloroform were added per ml of Trizol, shaken vigorously for 15-30 seconds and incubated at room temperature for 15 minutes. Samples were centrifuged for 15 minutes at 4˚C and 12,000 rcf. The aqueous phase was carefully collected in a separate tube. 500 μl isopropanol was added per 1 ml Trizol, inverted to mix and incubated at room temperature for 5-10 minutes. Samples were transferred to Qiagen RNeasy Spin Columns and RNA was isolated according to the manufacturer’s instructions.
Libraries were constructed using the Illumina TruSeq Stranded Total RNA Library Prep Kit.

tag: infection

value: IAV

tag: pb1 mutation site

value: PB1 KO mutation

tag: time

value: 6 days post-inoculation

value: lung


title: Adult_CTX_VIPcre_Dnmt3aWT_Sun1IP_H3K4me1_rep2

value: Cortex

value: Rabbit anti-H3K4me1, Abcam, Cat # ab8895


title: yeast unselected 40S 1-2

value: approximately 18 to 85

value: no


title: PR8;PB1-UFO_∆_3


title: Total 80S Ribosome FPs Hela cells 1 min Harringtonine [6_80S_NIH3T3_T2]

description:
MM_80S_Total_T2
processed data file: Master_Data_Table.xlsx

tag: rrna depletion

tag: library preparation kit

value: Takara

tag: index name

tag: index sequence

value: GAATTCGT


title: EGFP gene targeted by SpCas9:sgRNA targeting TGGC1

city: Raleigh

first: Chase

last: Beisel

state: NC

address: 840 Main Campus Drive

department: Chemical and Biomolecular Engineering

institute: North Carolina State University

description:
DC99
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

data_processing:
for samples "SpyPre", "SpyDbl", "SpyNoIPTG", "Spy105" were subjected to command line data processing. The grep function was used isolate the sequences making up the PAM library and discard the rest of the reads. The code requires that the neighboring 12nt of the protospacer match the reference sequence. The remaining sequences were sorted alphabetically and the code counts the number of times each sequence occurs in a given sample. A tab-delimited .txt file is then output.
sequencing reads were demuxed with trimmed adapters by the sequencing core facility for samples:  "DC64", "DC65", "DC67", "DC68", "DC69", "DC92", "DC93", "DC94", "DC97", "DC98", "DC99", and "DC100". The fastq files were input directly to the CRISPResso
Supplementary_files_format_and_content: "presed.txt" is a tab-delimited file containing the PAM sequences and corresponding read counts for the pre-sorted library. These PAM sequences were isolated from the fastq files meeting the sorting requirements (12nt matching the reference sequence upstream of the PAM)
Supplementary_files_format_and_content: "dblsed.txt" is a tab-delimited file containing the PAM sequences and corresponding read counts for the post-sorted library with two growth cycles. These PAM sequences were isolated from the fastq files meeting the sorting requirements (12nt matching the reference sequence upstream of the PAM)
Supplementary_files_format_and_content: "nosed.txt" is a tab-delimited file containing the PAM sequences and corresponding read counts for the post-sorted library grown in the presence of 0mM IPTG. These PAM sequences were isolated from the fastq files meeting the sorting requirements (12nt matching the reference sequence upstream of the PAM)
Supplementary_files_format_and_content: "105sed.txt" is a tab-delimited file containing the PAM sequences and corresponding read counts for the post-sorted library grown in the presence of 10mM IPTG. These PAM sequences were isolated from the fastq files meeting the sorting requirements (12nt matching the reference sequence upstream of the PAM)

source_name: U2OS gDNA

growth_protocol:
growth protocol for "SpyPre" samples: The PAM nucleotide library was freshly transformed into the host strain (already expressing dCas9 and the sgRNA) and recovered in LB media with antibiotics. The cells were grown, spun down, and washed in PBS three times prior to the next overnight growth in M9 base media. DNA was then isolated from a fraction of cells and saved for sequencing to determine the pre-selection library diversity.
growth protocol for "SpyDbl" samples: The PAM nucleotide library was freshly transformed into the host strain (already expressing dCas9 and the sgRNA) and recovered in LB media with antibiotics. The cells were grown, spun down, and washed in PBS three times prior to the next overnight growth in M9 base media. Following a growth cycles in M9 base media, the cells were then grown for two growth cycles in the selection media (M9 media with 0.002% glycerol, 0.02% cas amino acids,  0.2% xylose, and 0mM IPTG). The cells were then used for sequencing.
growth protocol for "SpyNoIPTG" samples: The PAM nucleotide library was freshly transformed into the host strain (already expressing dCas9 and the sgRNA) and recovered in LB media with antibiotics. The cells were grown, spun down, and washed in PBS three times prior to the next overnight growth in M9 base media. Following one growth cycle in M9 base media, the cells were then grown in the selection media (M9 media with 0.002% glycerol, 0.02% cas amino acids,  0.2% xylose, and 0mM IPTG). The cells were then used for sequencing.
growth protocol for "Spy105" samples: The PAM nucleotide library was freshly transformed into the host strain (already expressing dCas9 and the sgRNA) and recovered in LB media with antibiotics. The cells were grown, spun down, and washed in PBS three times prior to the next overnight growth in M9 base media. Following one growth cycle in M9 base media, the cells were then grown in the selection media (M9 media with 0.002% glycerol, 0.02% cas amino acids,  0.2% xylose, and 10mM IPTG). The cells were then used for sequencing.
growth protocol for "DC64", "DC65", "DC67", "DC68", "DC69", "DC92", "DC93", "DC94", "DC97", "DC98", "DC99", and "DC100": U2OS.EGFP cells were maintained in DMEM high glucose, 10% fetal bovine serum, and 1% penicillin and streptomycin, incubated at 37C with 5% CO2 using standard tissue culture techniques.

extract_protocol:
The reporter plasmid was isolated from pre- and post-xylose cultures (for samples "SpyPre", "SpyDbl", "SpyNoIPTG", and "Spy105") and DNA was amplified from this region with oligos containing the necessary Illumina adapters.
U2OS gDNA was extracted using the GeneJET Genomic DNA Extraction kit 72 hours after transfection. (Thermo Scientific Cat no K0721) following manufacturer's instruction. for "DC64", "DC65", "DC67", "DC68", "DC69", "DC92", "DC93", "DC94", "DC97", "DC98", "DC99", and "DC100"
The five nucleotide PAM library was inserted into the target plasmid backbone using Q5 mutagenesis.

treatment_protocol: treatment protocol for "DC64", "DC65", "DC67", "DC68", "DC69", "DC92", "DC93", "DC94", "DC97", "DC98", "DC99", and "DC100": Plasmid encoding the SpCas9 and sgRNA were prepped using Qiagen Endo-free Plasmid Maxi-Prep kit and transfected with the GenJet transfection reagent for U2OS cells version II (SignaGen). Cells were seeded at 200,000 cells / well in a 12 well dish one day prior to transfection. Cells were transfected for 72 hours.

tag: selection stage of 5n pam library -- pam screen

tag: number of growth cycles in d-xylose -- pam screen

tag: concentration of iptg in growth medium -- pam screen

tag: pam targeted by sgrna

value: spcas9 in egfp gene -- indel formation:TGGC1


title: PR8;PB1-UFO_∆_1

tag: genotype


title: Adult_CTX_VIPcre_Dnmt3aWT_Sun1IP_Input_rep1

tag: background


title: LS-MOUSE_VilarM05

tag: tissue

value: normal intestinal mucosa

tag: group

value: LS mouse model

value: control


title: PR8;PB1-UFO_SYN_3

value: Control


title: Adult_CTX_VIPcre_Dnmt3aWT_Sun1IP_H3K4me1_rep1

value: Vip-cre; Sun1; Dnmt3a +/+

tag: antibodies


title: EGFP gene targeted by SpCas9:sgRNA targeting CTGGG

description:
DC97
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:CTGGG


title: PR8;PB1-UFO_SYN_2

value: IAV


title: EGFP gene targeted by SpCas9:sgRNA targeting GTGGC2

description:
DC94
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:GTGGC2


title: EGFP gene targeted by SpCas9:sgRNA targeting CGGG

description:
DC98
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:CGGG


title: EGFP gene targeted by SpCas9:sgRNA targeting GCGGC

description:
DC92
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:GCGGC


title: EGFP gene targeted by SpCas9:sgRNA targeting TGGTG positive control

description:
DC69
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:TGGTG positive control


title: Adult_CTX_VIPcre_Dnmt3aWT_Sun1IP_H3K27ac_rep1

value: Cortex

value: Rabbit anti-H3K27ac, Abcam, Cat# ab4729


title: EGFP gene targeted by SpCas9:sgRNA targeting TCGG

description:
DC93
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:TCGG


title: EGFP gene targeted by SpCas9:sgRNA targeting CAGGG

description:
DC68
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:CAGGG


title: yeast eIF2beta bound 40S 1-3

tag: strain

value: SY194 (eIF2beta/SUI3-FLAG)

tag: rna fragment size selection

value: approximately 18 to 85

tag: co-ip

value: yes (anti-FLAG, Sigma A2220)

tag: protocol

value: RNA 40S footprint


title: yeast unselected 40S 1-1

value: SY182 (eIF3a/TIF32-FLAG)

value: no


title: Adult_CTX_VIPcre_Dnmt3aKO_Sun1IP_Input_rep1

value: Vip-cre; Sun1; Dnmt3a fl/fl


title: EGFP gene targeted by SpCas9:sgRNA targeting AAGT

description:
DC67
U2OS genomic DNA
no processed data file because fastq files were fed directly into CRISPResso

value: spcas9 in egfp gene -- indel formation:AAGT