title: k4_H5JLI


title: k4_HIRFD

status: Public on Sep 21 2023

type: SRA

city: Boston

first: Sylvan

middle: C

last: Baca

state: MA

address: 450 Brookline Ave

department: Center for Functional Cancer Epigenetics

country: USA

institute: Dana Farber Cancer Institute

library_source: genomic

data_processing:
cfChIP-seq / cfMeDIP-seq reads were aligned to the hg19 human genome build using the Burrows-Wheeler Aligner (BWA) version 0.7.1740
Non-uniquely mapping and redundant reads were discarded.
MACS v2.1.1.2014061641 was used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01.
Fragment locations were converted to bed files using bedtools (v2.29.2) bamtobed with the -bedpe flag set.
Assembly: GRCh37 - hg19

molecule: genomic DNA

organism: Homo sapiens

source_name: Breast

growth_protocol: All plasma extracted from whole blood by centrifugation for 10 min at 1500g at 4°

extract_protocol:
ChIP-seq libraries were prepped with ThruPLEX DNA-Seq Kit (Takara Bio, cat# R400675) following the manufacturer's instructions. After library amplification, the DNA was purified by AMPure XP (Beckman coulter, cat# A63880).
DNA was purified through Phenol extraction (Invitrogen, cat# 15593031) and the subsequent Ethanol precipitation with 3M NaOAc (Ambion, cat# AM9740) and glycogen (Ambion, cat# AM9510).

tag: cell type

value: Breast

tag: chip antibody


title: K4_ISIS32_1

source_name: Colorectal

value: Colorectal


title: K4_ISIS356_1

source_name: Ovarian

value: Ovarian


title: K4_ISIS13_1


title: K4_BYLU432_1

source_name: NSCLC

value: NSCLC


title: K4_CAI726_2


title: K4_ISIS109_1


title: k4_GFBVK


title: K4_BMS7_2

source_name: Renal

value: Renal


title: k4_L3V83


title: K4_BMS4_2


title: K4_BMS28_2


title: K4_ISIS337_1


title: K4_BMS35_2


title: K4_BMS31_2


title: k4_BMS15_2


title: K4_BMS19_2


title: k4_BMS12_2


title: K4_BELI82_2

source_name: Thymic

value: Thymic


title: K4_BELI92_2


title: k4_BELI90_2


title: K4_BELI70_2


title: K4_BELI83_2


title: K4_BELI64_2


title: K4_BELI74_2


title: K4_BELI117_2


title: K4_BELI68_2


title: K4_BELI56_2


title: K4_BELI123_2


title: K4_BCDC158_2


title: K4_AMP70_2


title: K4_BELI104_2


title: K4_AMP68_2


title: K4_AMP61_2


title: K4_AMP60_2


title: K4_AMP48_2


title: K4_BMS23_2


title: K4_AMP59_2


title: K4_AMP20_2


title: K4_AMP31_2


title: K4_AMP28_2


title: K4_AMP16_2


title: k4_BELI88_2


title: K4_BELI95_2


title: K4_5915


title: K4_AMP13_2


title: K4_5912


title: K27L8WWS_2


title: K27LXE8W_3


title: K27LXE8W_1


title: K4_4666


title: K27LXE8W_2


title: K27L8WWS_1


title: K27IGFSE_3


title: K4_AMP11_2


title: K27HU30E_2


title: K27H5UBF_1


title: K27IGFSE_1


title: K27IGFSE_2


title: K27HU30E_1


title: K27H5UBF_2


title: K27_TRHC88_2

source_name: Hepatocellular

value: Hepatocellular


title: K27_TRHC68_2


title: K27EZJFG_1


title: K27_TRHC82_2


title: K27_TRHC47_1


title: K27_TRHC25_2


title: K27_TRHC53_2


title: K27_TRHC66_2


title: K27_TRHC33_2


title: K27_TRHC238_2


title: K27_TRHC216_2


title: K27_TRHC161_2


title: K27_TRHC229_1


title: K27_TRHC192_1


title: K27L8WWS_3


title: K27_TRHC2


title: K27EZJFG_2


title: K27_TRHC169_2


title: k27_TRHC145_2


title: K27_TRHC123_2


title: K27_TRHC101_2


title: K27_TRC78_2

source_name: Prostate

value: Prostate


title: K27_TRC97_1


title: K27_TRHC103_2


title: K27_TRHC1


title: K27_TRC67_1


title: K27_TRC61_2


title: k27_TRHC131_2


title: K27_TRC42_1


title: K27_TRHC225_2


title: K27_TRC53_1


title: K27_TRC25_1


title: K27_TRHC208_2


title: K27_TRC23_1


title: K27_TRHC148_2


title: K27_TRC148_2


title: K27_TRC2


title: K27_TRC134_1


title: K27_TRC1


title: K27_TRC133_2


title: K27_TRC121_2


title: K27_TRC84_1


title: K27_TRC11_1


title: K27_SMAC70_2


title: K27_SMAC78_2


title: K27_SMAC64_2


title: K27_SMAC57_2


title: K27_SMAC49_2


title: K27_SMAC35_2


title: K27_SMAC11_2


title: K27_SMAC26_2


title: K27_SMAC05_2


title: K27_TRC30_1


title: K27_TRC40_1


title: K27_PS96_2


title: K27_PS75_2


title: K27_PS438_2


title: K27_PS55_2


title: K27_PS466_2


title: K27_PS475_2


title: K27_PS410_2


title: K27_TRC094_1


title: K27_PS325_2


title: K27_PS353_2


title: K27_PS381_2


title: k27_PS296_2


title: K27_PS219_2


title: K27_PS2


title: K27_PS197_2


title: k27_PS278_2


title: K27_PS154_1


title: K27_SMAC41_2


title: K27_SMAC23_2


title: K27_PS245_2


title: K27_PS1


title: K27_PS248_2


title: K27_PS123_2


title: K27_PS122_2


title: K27_PS173_1


title: K27_NEPC19

source_name: NEPC

value: NEPC


title: K27_NEPC21


title: K27_SATR1_2


title: K27_NEPC17


title: K27_NEPC15


title: K27_NEPC20


title: K27_NEPC14


title: K27_MGI958_1


title: K27_NEPC22


title: k27_MMYE1


title: K27_MGI956_2


title: K27_MGI954_1


title: K27_NEPC18


title: K27_MGI946_1


title: K27_MGI952_2


title: K27_MGI942_1


title: K27_MGI940_1


title: K27_MGI938_1


title: K27_MGI44_1


title: K27_Merk229

source_name: Merkel cell

value: Merkel cell


title: K27_Merk212


title: K27_Merk197


title: K27_Merk168


title: K27_Merk165


title: K27_LC46_CD

source_name: SCLC

value: SCLC


title: K27_LC46_Tak


title: K27_LC46_IDT


title: k27_LWZIR


title: k27_LC34


title: K27_NEPC16


title: k27_LC33m


title: k27_LC33g


title: k27_L3V83


title: k27_LC25


title: k27_LYCQK


title: k27_KVFUI


title: k27_KN7SQ


title: k27_JR6BR


title: K27_MGI933_1


title: K27_ISIS363_1


title: K27_ISIS356_1


title: K27_ISIS90_1


title: K27_ISIS342_1


title: K27_ISIS337_1


title: K27_ISIS32_1


title: k27_ISIS151_1


title: K27_ISIS21_1


title: K27_ISIS13_1


title: K27_HPC81_2


title: K27_ISIS109_1


title: K27_HPC81_1


title: k27_GFBVK


title: k27_HIRFD


title: K27_BYLU432_1


title: K27_BMS4_2


title: K27_BMS7_2


title: k27_KOL1X


title: K27_BMS35_2


title: K27_BMS23_2


title: K27_BMS28_2


title: K27_BMS31_2


title: k27_BMS15_2


title: K27_BMS19_2


title: k27_BMS12_2


title: K27_ISIS184_1


title: K27_BELI92_2


title: K27_BELI95_2


title: k27_BELI88_2


title: K27_BELI83_2


title: K27_BELI68_2


title: K27_BELI70_2


title: k27_BELI90_2


title: K27_BELI82_2


title: K27_BELI74_2


title: K27_BELI56_2


title: K27_CAI726_2


title: K27_BELI64_2


title: K27_BELI117_2


title: K27_BELI1


title: K27_BCDC158_2


title: K27_BELI123_2


title: K27_AMP68_2


title: k27_H5JLI


title: K27_AMP70_2


title: K27_AMP61_2


title: K27_AMP60_2


title: K27_AMP59_2


title: K27_AMP31_2


title: K27_AMP20_2


title: K27_AMP28_2


title: K27_BELI2


title: K27_AMP2


title: K27_AMP48_2


title: K27_AMP16_2


title: K27_AMP11_2


title: K27_AMP1


title: JS_PC_44_JS9177_S49


title: K27_AMP13_2


title: JS_PC_39_JS9177_S44


title: JS_PC_42_JS9177_S47


title: JS_PC_43_JS9177_S48


title: JS_PC_40_JS9177_S45


title: J_PCmlf32Me3


title: J_PCmlf31Me3


title: J_LCmlf007PanAc


title: J_LCmlf009Me3


title: J_LCmlf007Me3


title: J_LCmlf002PanAc


title: HP27557

source_name: Healthy

value: Healthy


title: J_LCmlf002Me3


title: HP31645Me3R1


title: J_HP030642Me3


title: J_HP030132Me3


title: HP140197


title: J_HP030132PanAc


title: HP19862


title: HP133814


title: HP119862


title: HP11717Me3R1v01


title: HP127557


title: HP135963


title: HP111616


title: J_PCmlf31PanAc


title: HP111016


title: HP10135963

data_processing:
cfChIP-seq / cfMeDIP-seq reads were aligned to the hg19 human genome build using the Burrows-Wheeler Aligner (BWA) version 0.7.1740
Non-uniquely mapping and redundant reads were discarded.
MACS v2.1.1.2014061641 was used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01.
Fragment locations were converted to bed files using bedtools (v2.29.2) bamtobed with the -bedpe flag set.
Assembly: GRCh37 - hg19
Library strategy: Low-pass whole genome sequencing (lpWGS)

extract_protocol:
Library was prepped by KAPA Hyper Prep Kit (KAPA Biosystems, cat# KK8500) according to the manufacturer’s protocol.
cell-free DNA (cfDNA) was extracted from plasma supernatant after cfCHIP by QIAmp Circulating Nucleic Acid kit (Qiagen, cat# 55114)

value: LPWGS


title: HP10132279


title: HP101286


title: HP10123027


title: HP10116305


title: HP10111717


title: HP10102703


title: HP10111016


title: HP28203Me3R1


title: HP10111616


title: HP101027


title: HP10095999


title: HP10075732


title: HP10077180


title: HP10059620


title: HP10095178


title: HP132279


title: HP100959


title: HP10069519


title: HP10058173


title: HP10059091


title: HP10133405


title: HP10047328


title: HP10053744


title: HP10038748


title: HP10033740


title: HP10036348


title: HP10045456


title: HP10035094_ACAGTG


title: HP10035094_CAGGCG


title: HP100337


title: HP10028203


title: HP090512


title: HP076550


title: HP098228


title: HP098228_H3K27Ac


title: HP10097407


title: HP075732


title: HP069519


title: HP059620


title: HP10041556


title: HP045456


title: HP053744


title: HP047328


title: HP044301


title: HP056703_H3K27Ac


title: HP041556_H3K27Ac


title: HP041556


title: HP038748_H3K27Ac


title: HP056703


title: K27_NEPC13


title: HP10031645


title: HP038748


title: HP034881_H3K27Ac


title: HP034881


title: HP036348


title: HP035094


title: HP030642_H3K27Ac


title: HP030642


title: HP030132_H3K27Ac


title: HP035094_H3K27Ac


title: HP024788


title: HP030132


title: HP030912


title: HP024732


title: HP026607


title: HP031645_H3K27Ac


title: hK4_PC053


title: gPC36_K4_3hr


title: gPC38K4


title: gPC18_K4_3hr


title: hK4_PC054


title: gPC38Ac


title: GPAcmel5

source_name: Melanoma

value: Melanoma


title: GPAcLC34


title: gLC5Ac


title: gLC6Ac


title: gPC18_K27ON


title: gLC4K4


title: gLC5K4


title: HP031645


title: gLC6K4


title: gLC46_K4_3hr


title: gLC47_K27ON


title: gLC46_K27ON


title: HP029376


title: gLC45_K4_3hr


title: gLC45_K27ON


title: gLC17K4


title: gLC17Ac


title: gLC16K4


title: GL10194K27R1

source_name: Glioma

value: Glioma


title: GL10192K4R2


title: GL10261K4R2


title: gLC16Ac


title: GL10083K27R1


title: GL10192K27R1


title: GL10040K4R2


title: GL10077K4R2


title: GL10025K4R2


title: GL10077K27R1


title: GL10040K27R1


title: GL10025K27R1


title: gLC47_K4_3hr


title: GL09923K27R1


title: GL09789K27R1


title: GL09923K4R2


title: GL09789K4R2


title: GL09828K27R1


title: GL09828K4R2


title: Gk4Pc59


title: GK4mel5


title: GK4mel6


title: GL10261K27R1


title: gK4_PC054


title: gK4_PC62_1


title: GK4Lc46


title: gK27_PC59b


title: GK4LC34


title: gK4_PC59a


title: gK27_PC62b


title: gK4_PC62a


title: GK4LC33


title: gHP034881K4


title: GeGENP8711AcR1


title: GAc2Lc46


title: gAcS_PC62


title: GAc2Lc47


title: GAc1H748


title: fNEPCmlf2M3R2


title: GAcPc59


title: gK4_PC62_2


title: fNEPCmlf2AcR1


title: fNEPCmlf1AcR1


title: EGSB4275

source_name: Esophageal

value: Esophageal


title: GK4Lc47


title: gHP034881Ac


title: EGS4488


title: fNEPCmlf1M3R2


title: gK4_PC053


title: EGS4352


title: EGS3867


title: EGS4300


title: EGS4325


title: EGS4348


title: EGS4275


title: EGS4206


title: EGS3808


title: EGS3906


title: FMNG0135_JS8585_S89


title: DgMe3_metPC_33_JS8753_S95


title: DgMe3_metPC_32_JS8753_S94


title: DgMe3_metPC_31_JS8753_S93


title: GAc1H348


title: EGS3801


title: EGS3794


title: DgMe3_metPC_34_JS8753_S96


title: EGS9920


title: DgMe3_metPC_26_JS8753_S89


title: DgMe3_metPC_22_JS8753_S86


title: DgMe3_metPC_29_JS8753_S92


title: DgMe3_metPC_28_JS8753_S91


title: DgMe3_metPC_23_JS8753_S87


title: DgM8mP28


title: DgM7mP27


title: DgM6mP26


title: EGS3845


title: DgM5mP24


title: DgM3mP34


title: DgM10mP31


title: DgM3mP22


title: D785K27R1


title: EGS3726


title: D756K27R1


title: DgMe3_metPC_24_JS8753_S88


title: D455K27R1


title: D196K27R1


title: CRpt070PanR2


title: CRpt257Me3R1


title: CRpt257bPanR2


title: D365K27R1


title: DgM9mP29


title: CRpt257PanR2


title: CRpt070Me3R1


title: CRpr043PanR2


title: COL9962


title: CRpr257Me3R1


title: CRpr749PanR2


title: COL9752


title: CRpr575PanR2


title: COL9629


title: COL9647


title: DgM4mP23


title: DgM3mP32


title: COL9614


title: DgM3mP33


title: COL9602


title: COL9517


title: COL9587


title: COL10387


title: COL10279


title: COL10380


title: COL10250


title: COL10241


title: COL10375


title: CG0052_JS8585_S86


title: COL10221


title: BC729PanAR2


title: COL10191


title: COL10194


title: COL10160


title: BC729K4R1


title: BC73934090M3R2


title: BC729K4R2


title: BC71846653M3R2


title: BC71846653AcR1


title: BC729K27R1


title: BC71843868M3R2


title: BC71841173M3R2


title: BC548K4R2


title: BC548K27R1


title: BC548K4R1


title: BC509PanAR2


title: BC509K4R2


title: BC40541PanAR2


title: BC509K4R1


title: BC509K27R1


title: BC40541K4R2


title: BC40541K4R1


title: BC40225PanAR2


title: BC73934348M3R2


title: BC40541K27R1


title: BC40225K4R2


title: BC40225K4R1


title: BC40088K4R1


title: BC40088K27R1


title: BC40033K4R2


title: BC71838950M3R2


title: BC40033PanAR2


title: BC40088PanAR2


title: BC548PanAR2


title: BC39103K4R1


title: BC40225K27R1


title: BC40033K27R1


title: BC39103PanAR2


title: BC39103K4R2


title: BC39103K27R1


title: BC320K4R2


title: BC320PanAR2


title: BC287PanAR2


title: BC320K4R1


title: BC320K27R1


title: BC287K4R1


title: BC287K27R1


title: advPC8


title: advPC20


title: advPC3


title: BC40088K4R2


title: advPC18


title: advPC_9_JS8585_S71


title: BC40033K4R1


title: advPC_3_JS8584_S121


title: advPC_20_JS8584_S124


title: advPC_21_JS8585_S75


title: advPC_18_JS8585_S96


title: Ac_mPC18_all


title: Ac_mPC17_3R


title: Ac_mPC15_all


title: Ac_mPC15_3R


title: BC287K4R2


title: Ac_mPC16_1R


title: advPC_13_JS8585_S72


title: Ac_mPC18_1R


title: Ac_mPC18_3R


title: advPC_3_JS8585_S94


title: Col-0 seedling Ribo-seq sample 5

status: Public on Sep 20 2023

city: East Lansing

first: Polly

last: Hsu

state: MI

address: 603 Wilson Road

department: Biochemistry and Molecular Biology

institute: Michigan State University

description:
Root Ribo-seq sample 5
ribosome-protected mRNA fragment

library_source: transcriptomic

data_processing:
For Ribo-seq libraries, adaptor (AGATCGGAAGAGCACACGTCT) was clipped with fastx_clipper (FASTX toolkit v0.0.14).
For both RNA-seq and Ribo-seq, we used Bowtie2 (v2.3.4.1) to remove the r/t/sn/sno sequences.
To calculate the translation efficiency, both RNA-seq and Ribo-seq reads were mapped to the CDS region of the transcriptome with STAR aligner (RNA-seq parameters: --outFilterMismatchNmax 2 --outFilterMultimapNmax 20 --outFilterType BySJout --alignSJoverhangMin 8 --alignSJDBoverhangMin 2; Ribo-seq used the same parameter except --outFilterMismatchNmax 1).
The resulting bam files were merged and  used for quantifying the TPM (transcript per million) of each gene with RSEM (v1.3.1).
Genome_build: Araport11
Supplementary_files_format_and_content: RSEM output txt files for Ribo-seq and RNA-seq

molecule: total RNA

organism: Arabidopsis thaliana

source_name: 7-day old Seedlings

growth_protocol: Arabidopsis Col seeds were surface sterilized with 70% ethanol for 5 minutes, followed by 33% bleach and 0.03% Tween 20 for 10 minutes, and rinsed with sterile water 5 times. The seeds were imbibed at 4°C in dark for 2 days, and then grown hydroponically with sterile liquid media (2.15 g/L Murashige and Skoog salt, 1% sucrose, 0.5 g/L MES, pH 5.7) shaken at 85 rpm under 16 hours light and 8 hours dark at 22 °C for one week.

extract_protocol:
Plant lysates were prepared as previously described. Briefly, per 0.1 g of grounded tissue power was resuspend in 400 µL of lysis buffer (100 mM Tris-HCl [pH 8], 40 mM KCl, 20 mM MgCl2, 2% [v/v] polyoxyethylene [10] tridecyl ether [Sigma, P2393], 1% [w/v] sodium deoxycholate [Sigma, D6750], 1 mM dithiothreitol, 100 µg/mL cycloheximide [Sigma, C4859], 100 µg/mL chloramphenicol [Sigma R4408], and 10 units/mL DNase I [Epicenter, D9905K]). The lysates were spined at 3,000 g for 3 min and the supernantant was transferred to new tube and subsequently centrifuged at 20,000 g for 10 min. The supernantant was transferred to new tube and the RNA concentration was determined using Qubit RNA HS assay (Thermo Fisher Scientific; Q32852). Aliquots of 100 µL and 200 µL of the lysates were made and flash frozen in liquid nitrogen and stored in -80°C freezer until further process
Ribo-seq library construction: Ribosome footprints were obtained using 200 µL of the lysates above and the sequencing libraries were constructed following our previous method with the following modifications: briefly, after RNase I digestion (50 unit nuclease per 40 µg of RNA, the nuclease was provided in TruSeq Mammalian Ribo Profile Kit, illumina, RPHMR12126) and size exclusion column step, RNA > 17 nt was isolated by RNA Clean & Concentrator-25 kit (Zymo Research, R1017) and separated in 15% Urea-TBE gels (Invitrogen, EC68852BOX). Gel slices between 28-30 nt were isolated and the RNAs were purified as previously described. Next, rRNA depletion was performed using RiboZero Plant Leaf kit (Illumina, MRZPL1224) with one quarter of recommended reaction volume. Ribo-seq libraries were then constructed using TruSeq Mammalian Ribo Profile Kit (illumina, RPHMR12126) as previously described with 9 cycles of PCR amplification. Equal molarity of libraries were pooled and sequenced on Hi-Seq 4000 using single end 50 bp sequencing. RNA-seq library construction: Total RNA greater than 200 nt was purified using 100 µL of the lysates above as previously described with RNA Clean & Concentrator-25 kit (Zymo Research, R1017). RNA integrity was evaluated using Bioanalyzer (Agilent) RNA pico chip and RNA integrity number (RIN) ranging from 7.2 to 7.7 were observed among the samples. A 4 µg of RNA per sample was subjected to rRNA depletion using RiboZero Plant Leaf kit (Illumina, MRZPL1224) following the manufacturer’s recommendation. Then, 100 ng of rRNA-depleted RNA was fragmented to around 200-nt long based on the RIN reported by Bioanalyzer, and strand-specific sequencing libraries were made using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7760S) with 8 cycles of amplification. Equal molarity of libraries were pooled and sequenced on Hi-Seq 4000 using pair-end 100 bp sequencing.

treatment_protocol: At Zeitgeber time 3 (3 hours after light on), DMSO that corresponded to 0.1% of media volume was added to the media (these were mock samples of our large-scale experiment). After 20 and 60 minutes later, three biological replicates of each time point were harvested and immediately flash frozen in liquid nitrogen.

tag: organ

value: Seedlings

tag: cultivar


title: Col-0 seedling Ribo-seq sample 6

description:
Root Ribo-seq sample 6
ribosome-protected mRNA fragment


title: BraKO_d5_rep2 (ATAC-seq)

status: Public on Jul 26 2023

city: Freiburg im Breisgau

first: Sebastian

last: Arnold

address: Albertstraße 25

department: Institute of Experimental and Clinical Pharmacology and Toxicology

country: Germany

institute: University Freiburg

data_processing:
For RNA-seq ESCs: Low-quality bases and adapter-containing reads were trimmed using Galaxy platform Trim Galore (Galaxy Version 0.4.3.1) and reads were aligned to the mouse reference genome mm10 (mm10_UCSC_07_15, RNA STAR Galaxy Version 2.7.2b) with default parameters. Duplicates were removed with RmDup (Galaxy version 2.0.1) and genomic features were counted using htseq-count (Galaxy Version 0.9.1) with following settings: mode-union, stranded-no, minimum alignment quality-10 and feature type-exon. Differential expression analysis was carried out using DESeq2 (Galaxy Version 2.11.40.6+galaxy1) with subsequent filtering for adjusted p-value<0.05 and log2FC as indicated in the figure legends.
For RNA-seq embryos: Reads were mapped to and annotated by using the mouse reference genome GRCm38/mm10 (iGenomes, Illumina; archive-2015-07-17-32-40 and archive-2015-07-17-33-26 for GRCM38 and mm10, respectively) containing the chromosomes 1-19, X, Y and M using Rsubread v1.28.1 package in R v3.4.3 (Liao et al., 2013). Genomic features were counted using the Rsubread::featureCounts function (Liao et al., 2013). Differential expression analysis was carried out using DESeq2 v1.18.1 in R (Love et al., 2014), which applies Negative Binomial GLM fitting and Wald statistics on the count data, with subsequent filtering for adjusted p-value<0.05 and log2FC as indicated in the figure legends. Prior to fitting and statistical analysis, counts were normalized by library size (DESeq2::estimateSizeFactors) and gene-wise dispersion (DESeq2::estimateDispersions) to normalize the gene counts by gene-wise geometric mean over samples.
For ChIP-seq: Low-quality bases and adapter-containing reads were trimmed using Galaxy platform Trim Galore (Galaxy Version 0.4.3.1). Reads were mapped to the mm10 genome using Galaxy platform Bowtie2 v2.3.4.3+galaxy0) (Langmead and Salzberg, 2012) with default settings and the duplicates removed with RmDup (Galaxy version 2.0.1). Peaks were detected using MACS2 (v2.1.1.20160309.6, Zhang et al., 2008) with BAMPE format of the input file, default settings for building the shifting model (confidence enrichment ratio against background 5-50; band width 300) and minimum q-value cut-off for detection of 0.01. Peaks were called for each individual replicate and for both replicates together. Intersection (minimal overlap of at least 1 bp) of the called peaks using bedtools intersect intervals (Galaxy Version 2.29.2) were defined as high confidence peaks and used for further analysis. Differential binding of EoGFP and BraGFP was analysed by using the Galaxy DiffBind tool (v2.10.0) with default settings and a minimal FDR threshold of 0.01 and subsequent filtering for minimal fold change (FC) of 2.5 (Galaxy version 1.1.1)
For ATAC-seq: Low-quality bases and adapter-containing reads were trimmed using Galaxy platform Trim Galore (Galaxy Version 0.4.3.1). Reads were mapped to the mm10 genome using Galaxy platform Bowtie2 v2.3.4.3+galaxy0) (Langmead and Salzberg, 2012) with default settings and the duplicates removed with RmDup (Galaxy version 2.0.1). Differential accessibility analyses were performed using DiffBind tool (Galaxy Version 2.10.0) with default settings and FDR threshold of 0.01 and subsequent filtering for minimal fold change (FC) of 2.5 (Galaxy version 1.1.1).
Coverage: For the generation of Coverage files for RNA-seq, ATAC-seq and ChIP-seq analysis corresponding bam files from biological replicates were merged using Merge BAM files (Galaxy version 1.2.0), the Coverage files were created using bamCoverage tool (Galaxy version 3.3.2.0.0) bin size 10 bases and normalization to RPKM.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Coverage files in BigWig format for RNA-seq, ChIP-seq and ATAC-seq reads are normalized to RPKM. Bed files for EoGFP and BraGFP ChIP-seq unique and common peaks contain coordinates of the peaks. Bedfiles for Diffbind analysis ATAC BraKO versus EoKO contain coordinates of the peaks with FDR threshold 0.01. Processed ESC RNA-seq results showing differential gene expression between WT and BraKO in tab-delimited txt format. Processed embryo RNA-seq results show raw count tables per replicate in tab-delimited txt format.

organism: Mus musculus

source_name: 5 days BraKO embryoid bodies (ActA)

growth_protocol: A2lox.Cre Mouse ESCs were grown in standard serum containing conditions (DMEM supplemented with 15% ESC-grade FBS, 2mM L-Glutamine, 50U/ml Penicillin/Streptomycin, 1xNEAA, 1mM sodium pyruvate, 100µM β-mercaptoethanol) with 1000 U/ml LIF, 3 mM CHIR99021 and 1 mM PD0325901 on amonolayer of mitotically inactivated STO feeder cells (SNL76/7). For embryoid body formation 1x10^5 feeder-depleted ESCs (WT and BraKO) were plated in 4 ml ESGRO Complete Basal medium in 60 mm non-adhesive dishes. Medium was changed on d2 and d5 using 4 ml ESGO Complete Basal medium supplemented with 30ng/ml human recombinant ActivinA (ActA, R&D systems).

extract_protocol:
The protocol was modified from Kaestner Lab, 2020. In brief, EBs at day 5 of differentiation were trypsinised to single-cell suspension, washed with PBS and 50.000 cells were lysed in 50 µl ATAC-seq lysis buffer. Nuclei were mixed with 50 µl of transposition reaction mix containing Tagment DNA Enzyme 1 (Nextera DNA Library Preparation Kit, Illumina). The transposition reaction was carried out at 37°C and 600rpm for 30 min, purified using the Qiagen MinElute Kit and eluted in EB-buffer (Qiagen).
DNA was amplified by PCR with an appropriate number of PCR cycles (5-8) using Custom Nextera PCR primer and the NEBNext High-Fidelity 2x PCR Master Mix (Nextera DNA Library Preparation Kit (New England BioLabs, E7530L) and purified with the Qiagen MinElute Kit (Qiagen, 28004). The libraries were size-selected using SPRI select beads (Beckman Coulter) to obtain fragments between <1000 bp of size. DNA concentration was measured with a Qubit Fluorometer (Thermo Fischer, Q32854) and fragment size determined with a Bioanalyzer (Agilent).

treatment_protocol: Embryoid bodies (EBs) were treated with 30ng/ml human recombinant Activin A from day 2 to 5.

value: embryoid bodies

tag: genotype

value: BraKO

tag: day

tag: treatment

value: ActA


title: advPC_18_JS8584_S123


title: Ac_mPC17_all


title: WT_d5_rep3 (RNA-seq)

source_name: 5 days A2lox.Cre embryoid bodies (ActA)

growth_protocol: A2lox.Cre Mouse ESCs were grown in standard serum containing conditions (DMEM supplemented with 15% ESC-grade FBS, 2mM L-Glutamine, 50U/ml Penicillin/Streptomycin, 1xNEAA, 1mM sodium pyruvate, 100µM β-mercaptoethanol) with 1000 U/ml LIF, 3 mM CHIR99021 and 1 mM PD0325901 on amonolayer of mitotically inactivated STO feeder cells (SNL76/7). For embryoid body formation 200 feeder-depleted ESCs were plated in 40µl ESGRO Complete Basal medium in ultra-low attachment 96-well plates for 2 days. On day 2, 48 EBs were transferred to 60 mm non-adhesive dishes in 4 ml ESGO Complete Basal medium supplemented with 30ng/ml human recombinant ActivinA (ActA, R&D systems). On day 4, medium supplemented with ActA was changed. Cells were harvested on day 5.

extract_protocol:
RNeasy Mini Kit (Qiagen) with on-column DNase digest.
NGS library preparation was performed using the Ultra RNA Library Prep Kit.

value: WT


title: Col-0 seedling Ribo-seq sample 4

description:
Root Ribo-seq sample 4
ribosome-protected mRNA fragment


title: BraKO_d5_rep2 (RNA-seq)


title: BraKO_d5_rep1 (RNA-seq)


title: BraKO+BraGFP_rep2 (ChIP-seq)

source_name: 5 days BraKO+BraGFP embryoid bodies (ActA+Dox)

growth_protocol: A2lox.Cre Mouse ESCs were grown in standard serum containing conditions (DMEM supplemented with 15% ESC-grade FBS, 2mM L-Glutamine, 50U/ml Penicillin/Streptomycin, 1xNEAA, 1mM sodium pyruvate, 100µM β-mercaptoethanol) with 1000 U/ml LIF, 3 mM CHIR99021 and 1 mM PD0325901 on amonolayer of mitotically inactivated STO feeder cells (SNL76/7). For embryoid body formation 2.5x10^5 feeder-depleted ESCs (EoKO+EoGFP or BraKO+BraGFP) were plated in 10 ml ESGRO Complete Basal medium in 100 mm non-adhesive dishes. Medium was changed on d2 and d5 using 10 ml ESGO Complete Basal medium supplemented with 30ng/ml human recombinant ActivinA (ActA, R&D systems).

extract_protocol:
ChIP seq was done as described previously (Tosic et al., 2019). Briefly, on day 5 of differentiation, EBs were trypsinised and approximately 3x10^7 cells was used per ChIP. Cells were double cross-linked using 2 mM disuccinimidyl glutarate followed by 1% formaldehyde. Chromatin was isolated, sonicated and incubated with 5µg anti-GFP antibody (Abcam, ab290) coupled to ProteinG beads overnight at 4°C. The next day, beads were washed on ice with RIPA buffer and TBS. DNA was eluted, de-crosslinked and purified.
Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S), size selected using the SPRI select beads (Beckman Coulter) to obtain fragments between 300-600 bp of size.

value: ActA+Dox


title: Col-0 seedling Ribo-seq sample 3

description:
Root Ribo-seq sample 3
ribosome-protected mRNA fragment


title: EoKO+EoGFP_rep2 (ChIP-seq)

source_name: 5 days EoKO+EoGFP embryoid bodies (ActA+Dox)


title: EoKO+EoGFP_rep1 (ChIP-seq)


title: Col-0 seedling Ribo-seq sample 2

description:
Root Ribo-seq sample 2
ribosome-protected mRNA fragment


title: Col-0 seedling Ribo-seq sample 1

description:
Root Ribo-seq sample 1
ribosome-protected mRNA fragment


title: BraKO_d5_rep1 (ATAC-seq)


title: EomesBraΔEpi E7.5_embryo_rep2 (RNA-seq)

description: EomesBraΔEpi

source_name: E7.5 EomesBraΔEpi mouse epiblast, strain background: mixed 129SvEv/C57Bl/6

extract_protocol:
RNA from E7.5 mouse epiblasts was extracted with Qiagen RNeasy Micro kit with on-column DNase digest.
Libraries were prepared from 10 ng of RNA with the Ovation SoLo RNA-seq Systems kit (NuGEN).

treatment_protocol: No treatment.

tag: strain

value: mixed 129SvEv/C57Bl/6

value: epiblast

value: EomesBra<delta>Epi


title: WT_d5_rep2 (RNA-seq)


title: 215H Wagyu

status: Public on Jul 01 2023

city: Cleveland

first: Fulai

last: Jin

state: Ohio

address: 10900 Euclid Avenue  Biomedical Research Building

department: Department of Genetics and Genome Sciences

institute: Case Western Reserve University, Cleveland

description: 10x Genomics

library_source: transcriptomic single cell

data_processing:
Raw sequencing data were processed using Cell Ranger 6.0.0 (10X Genomics) and ARS-UCD1.2 bovine genome.
Processed data were analyzed using Seurat (version 4.0.4) in R.
Assembly: ARS-UCD1.2 bovine genome
Supplementary files format and content: cattle_scRNA_10X normalized data matrix
Supplementary files format and content: Complete_10x_cattle_Metadata.csv includes the cell annotation information for the Complete_10x_cattle_normalized_counts_data.csv file.

organism: Bos taurus

source_name: skeletal muscle

extract_protocol:
Muscle tissue was rinsed with PBS and minced with a dissecting scissors. Minced muscle tissue was then digested in Dulbecco’s Modified Eagle’s medium (DMEM) contaning 0.75 U/ml collagenase D and 1 U/ml of Dispase II for 1 h at 37°C. The tissue slurry was filtered through a 100 µm cell strainer and pelleted through centrifugation. For 10x Genomics scRNAseq, debris and dead cells were removed from isolated cells using a debris removal solution (130-109-398) and a dead cell removal kit (130-090-101) from Miltenyi Biotec, respectively.
scRNAseq were performed using cells isolated from bovine muscle biopsy samples (2 animals of each breed). Libraries were constructed using Chromium Single Cell 3' Reagent Kits v3 (10x Genomics). The cDNA libraries were sequenced on the Illumina Hi-seq platform using 150 bp paired-end sequencing at a depth of more than 20,000 read pairs/cell.

tag: tissue

value: skeletal muscle

tag: breed

value: Wagyu

value: skeletal muscle tissue cells

tag: age

value: 4-month old


title: 204H Crossbred

description: 10x Genomics

value: Crossbred


title: BraKO_d5_rep3 (RNA-seq)


title: WT_d5_rep1 (RNA-seq)


title: advPC_17_JS8585_S73

value: Prostate


title: BraKO+BraGFP_rep1 (ChIP-seq)


title: RNA Il17eGFP_d3_npTh17_all_3

status: Public on Jun 01 2023

city: Cambridge

first: Pratiksha

middle: I

last: Thakore

address: 415 Main St

institute: Broad Institute

description: RNA_seq_npTh17_pTh17_Il17eGFP_rawcounts.txt

data_processing:
Base-calling was performed by RTA v2.4.11, and the BCL files were translated to fastqs using bcl2fastq2 v2.20.0.
RNA-seq: Paired reads were mapped to mouse annotation mm10 using TopHat, and after duplicates were removed, counts tables were generaed using htseq counts. For more details, see Method section of paper, "Bulk RNA-seq pre-processing"
Assembly: mm10
Supplementary files format and content: RNA-seq: comma-separated values (CSV) files include read count values for each gene in each sample.

source_name: Sorted live cells polarized in npTh17 conditions for 72 hours

growth_protocol: For the in vitro differentiation of naïve CD4+ T cells, naïve CD4+ CD44- CD62L+ cells were activated with plate-bound anti-CD3 and anti-CD28 antibodies in the presence of cytokines. For the acquisition of ex vivo Th17 cells, mice were immunized subcutaneously into the flanks with an emulsion of the MOG35-55 peptide and M. tuberculosis H37Ra extract in CFA for the induction of EAE.

extract_protocol:
6,000 cells were sorted by FACS from the in vitro culture or draining lymph nodes and central nervous system of EAE mice into 5ul TCL buffer supplemented with 1% 2-mercaptoethanol and stored at -80°C until library preparation.
For bulk RNA-seq, full-length RNA-seq libraries were prepared following a modified SMART-Seq2 protocol as previously described. The cDNA concentration was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and normalized to 0.25ng/ul. The cDNA libraries were prepared using the Nextera XT DNA Library Preparation kit (Illumina). Final libraries were confirmed to have a size of 500 bp using a Bioanalyzer (Agilent). Prior to sequencing, the uniquely barcoded libraries were pooled, normalized to 2 nM, and denatured using 0.1 N NaOH. Flow cell cluster amplification and sequencing were performed according to the manufacturer’s protocols using the paired-end Illumina sequencing (38bp X 2) with 75 cycle Nextseq 500 high output V2 kit (Illumina).

value: Spleen and lymph nodes

value: Wildtype


title: EomesBraΔEpi E7.5_embryo_rep4 (RNA-seq)

description: EomesBraΔEpi


title: EomesBraΔEpi E7.5_embryo_rep3 (RNA-seq)

description: EomesBraΔEpi


title: RNA Il17eGFP_d3_npTh17_all_2


title: EomesBraΔEpi E7.5_embryo_rep1 (RNA-seq)

description: EomesBraΔEpi


title: 214H Brahman

description: 10x Genomics

value: Brahman


title: 216H Crossbred

description: 10x Genomics


title: RNA Il17eGFP_d2_npTh17_GFP_2

source_name: Sorted GFP+ cells polarized in npTh17 conditions for 48 hours


title: 209H Brahman

description: 10x Genomics


title: RNA Il17eGFP_d2_npTh17_GFP_3


title: RNA Il17eGFP_d2_npTh17_GFP_1


title: RNA Il17eGFP_d3_npTh17_GFP_2

source_name: Sorted GFP+ cells polarized in npTh17 conditions for 72 hours


title: RNA Il17eGFP_d3_npTh17_GFP_3


title: 208H Wagyu

description: 10x Genomics


title: RNA Il17eGFP_d2_pTh17_all_1

source_name: Sorted live cells polarized in pTh17 conditions for 48 hours


title: RNA Il17eGFP_d3_pTh17_all_3

source_name: Sorted live cells polarized in pTh17 conditions for 72 hours


title: RNA Il17eGFP_d3_npTh17_GFP_1


title: RNA Il17eGFP_d3_pTh17_all_1


title: RNA Il17eGFP_d3_npTh17_all_1


title: RAD21 ChIP-M of wt stembryos at 96h, rep1

status: Public on Apr 28 2023

city: Lausanne

first: Lucille

last: Lopez-Delisle

address: Station 19

country: Switzerland

institute: EPFL

data_processing:
All scripts are available on https://github.com/lldelisle/scriptsForRekaikEtAl2022
Fastqs of the 3 replicates of wt_144h_pSer2PolII were concatenated before any processing.
Adapter sequences were trimmed from reads using cutadapt (Martin et al 2011) version 1.16 (-a 'GATCGGAAGAGCACACGTCTGAACTCCAGTCAC' -A 'GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT' for ChIP and -a 'CTGTCTCTTATACACATCTCCGAGCCCACGAGAC' -A 'CTGTCTCTTATACACATCTGACGCTGCCGACGA' for ChIPmentation).
Trimmed reads were mapped on mm10 genome or the mutant genome Ins(2xCBS-d4d8) for the corresponding  mutant CTCF ChIPmentation) using bowtie2 version 2.3.4.1 (Langmead et al. 2012).
Only aligments with proper pairs and mapping quality above 30 were kept using samtools 1.8.
Peaks and coverage were obtained with macs2 version 2.1.1.20160309 (--format BAMPE --gsize 1870000000 --call-summits --bdg).
In order to better compare ChIP, ChIP for the same protein with a time-course were normalized together using a custom python script (available in the github repository). Similarly, this normalization was used when mutant and control were performed in parallel (see supplementary table).
For H3K27ac and H3K27me3, in figure 7B and S10A, in order to reduce the impact of variations in gastruloid growth speed, the profile of mutant and corresponding wild-type were corrected using the time-course experiment and the profile around the HoxA cluster as a guide (see github repository for more details).
Assembly: mm10 or Ins(2xCBS-d4d8) for the sample Ins(2xCBS-d4d8)_144h_CTCF
Supplementary files format and content: *.narrowPeak.gz: narrowPeak from macs2
Supplementary files format and content: *.bigwig: macs2 coverage
Supplementary files format and content: *_Normalized.bigwig: macs2 coverage normalized between experiments for time-course or mutant and corresponding controls (see supplementary table)
Supplementary files format and content: *_Normalized_HCN_*h.bedgraph.gz: macs2 normalized coverage on the Hox clusters corrected for variation in gastroloid growth speeds using the profile around HoxA cluster and time-course as a guide

source_name: stembryo

extract_protocol:
Collected stembryos were pooled in a 15 ml falcon tube, washed with PBS and resuspended in 1 ml PBS containing 1% formaldehyde for fixation during 10 min at room temperature. The crosslink reaction was stopped by adding a glycine solution to a final concentration of 0.125 M. Fixed stembryos were pelleted and stored at -80°C until further use.
ChIP and ChIP-M experiments were performed according to the protocol described in (Rodriguez-Carballo et al., 2017) and (Darbellay et al., 2019), and adapted for stembryos samples. Samples were resuspended in a sonication buffer (Tris HCl pH=8.0 50 mM; EDTA 10 mM; SDS 0.25% and protease inhibitors) and sonicated in a Covaris E220 device for 14 min (duty cycle 2%, peak incident power 105 W) to obtain an average chromatin fragment size of 300-500 bp. A dilution buffer (HEPES pH=7.3 20 mM; EDTA 1 mM; NP40 0.1%; NaCl 150 mM and protease inhibitors) was added to the sonicated chromatin and incubated with the antibody-bead complex (Pierce Protein A/G Magnetic Beads, Thermo Scientific) overnight at 4°C. Sequential washes were then performed twice with RIPA buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 140 mM and protease inhibitors), RIPA High salt buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; Sodium Deoxycholate 0.1% TritonX-100 1%; NaCl 500 mM and protease inhibitors), LiCl buffer (Tris HCl pH=8.0 10 mM; EDTA 1 mM; LiCl 250 mM; Sodium Deoxycholate 0.5%; NP40 0.5% and protease inhibitors) and Tris HCl buffer (pH=8.0 10mM and protease inhibitors). For ChIP experiments (H3K27ac (ab4729, Abcam), H3K27me3 (39155, Active Motif), CDX2 (sc-393572, Santa Cruz) and PolII-Ser2p (04-1571, Millipore)), DNA fragments were incubated in the elution buffer (Tris HCl pH=8.0 10 mM; EDTA 5 mM; NaCl 300 mM and SDS 0.1%) containing proteinase K and purified using the Qiagen MiniElute kit. A phosphatase inhibitor (PhosStop, Roche) was used during PolII-Ser2p ChIP incubation and beads wash. The DNA library was produced using TruSeq adapters and amplified with the Kapa HiFi library kit (Roche) using a number of cycles determined by qPCR. For ChIP-M samples (CTCF (61311, Active Motif), PolII (ab817, Abcam), RAD21 (ab992, Abcam) and NIPBL (A301-779A, Bethyl Laboratories)), DNA fragments bound to the antibody-bead complex were tegmented using the Nextera Tegmentation kit. Beads were resuspended in the tegmentation buffer and incubated at 37° for 2 min with 1 µl of the Tn5 transposase. Fragment were then eluted and purified as described previously, and amplified using Nextera primers. Final DNA libraries were purified and size selected using AMPure XP magnetic beads (Beckman Coulter), and a fragment analysis was performed before sequencing

value: stembryo

value: mES cells

value: wt

tag: stage (in_hours_after_aggregation)

value: RAD21 (ab992, Abcam)


title: RNA Il17eGFP_d2_pTh17_all_3


title: TCH6_R

status: Public on Apr 27 2023

city: Houston

first: Pavel

last: Sumazin

state: TX

address: 1102 Bates St

department: Pediatric

institute: Baylor College of Medicine

data_processing:
RNA-Seq reads were aligned and quantified using RSEM (version 1.30) (PMID: 21816040) and Bowtie2 (version 2.10) (PMID: 22388286) to human reference GRCh38 {(GENCODE v32/Ensembl 98)(refdata-gex-GRCh38-2020-A dated 2020-07-07 downloaded from 10X Genomics with GENCODE gtf dated 2019-09-05)}.
Assembly: hg19
Supplementary files format and content: tab-delimited text files include count values for each Sample

source_name: marrow

extract_protocol:
RNA was extracted using the Qiagen RNeasy Plus mini kit with a genomic DNA elimination column (74104).
Extracted RNA samples were profiled using Illumina Novaseq 600 with 25M reads per sample.
Briefly, RNA quality was confirmed, 150bp paired-end mRNA libraries were prepared, and libraries were sequenced at a depth of 25M reads per sample on the NovaSeq 6000 platform (Illumina).

value: marrow

tag: Sex

value: M

tag: diagnosis

value: pediatric AML

tag: timepoint

value: Relapse


title: RNA-seq of wt stembryos at 144h, Time-course_rep1

data_processing:
All scripts are available on https://github.com/lldelisle/scriptsForRekaikEtAl2022
Adapter sequences were trimmed from reads using cutadapt (Martin et al 2011) version 1.16 (-a 'GATCGGAAGAGCACACGTCTGAACTCCAGTCAC' -A 'GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT').
Trimmed reads were aligned on mm10 with STAR version 2.7.7a (Dobin et al. 2013) with ENCODE options using a custom gtf based on Ensembl version 102 (https://doi.org/10.5281/zenodo.4596490).
Only uniquely mapped reads (tag NH:i:1) were kept using bamFilter version 2.4.1 (Barnett et al. 2011).
FPKM values were computed using cufflinks version 2.2.1 (Trapnell et al. 2010; Roberts et al. 2011) with default parameters.
Assembly: mm10
Supplementary files format and content: *_FPKM.txt.gz: output of cufflinks with FPKM values per gene
Supplementary files format and content: all_FPKM.txt.gz: all FPKM values combined into a single table, one raw per gene_id and one column per sample

extract_protocol:
Stembryos for each condition were collected and pooled in a 2 ml Eppendorf tube. After centrifugation and medium removal, pelleted stembryos were stored at -80°C until RNA extraction.
RNeasy Plus Micro kit (Qiagen) with on-column DNase digestion was used for RNA extraction following the manufacturer’s instructions.
RNA-seq library preparation with Poly-A selection was performed with 1 µg of purified RNA using the TruSeq Stranded mRNA kit from Illumina and following the manufacturer’s protocol. Library quality was assessed with Fragment analyzer.


title: RNA-seq of wt stembryos at 144h, Time-course_rep2


title: RNA Il17eGFP_d3_pTh17_all_2


title: TCH6_D

value: Diagnosis


title: RNA-seq of wt stembryos at 120h, Time-course_rep2


title: TCH5_R


title: TCH5_D


title: TCH4_D

value: F


title: TCH4_R


title: TCH2_R


title: TCH3_R


title: TCH2_D


title: TCH1_D


title: RNA-Seq 35S:mim156, 27 C, rep2

status: Public on Feb 11 2023

city: Riverside

first: Meng

last: Chen

state: CA

address: 2150 Batchelor Hall, 900 University Ave.

institute: University of California, Riverside

description: 35S:mim156, 27 C, rep2

data_processing:
RNA-seq data analysis was performed using the homemade pRNASeqTools pipeline (https://github.com/grubbybio/pRNASeqTools). The raw reads were mapped to the Araport11 genome using STAR version 2.7.7a.
The number of reads mapped uniquely to each annotated gene was counted using FeatureCounts version 2.0.1.
Transcript levels were measured in fragments per kilobase per million total mapped fragments (FPKM). Differentially expressed genes were identified using DEseq2 v1.35.0 with a fold change of two and p < 0.01.
Assembly: Araport11
Supplementary files format and content: An Excel file includes mRNA abundance data (FPKM) for all samples.

molecule: polyA RNA

source_name: whole seedling

growth_protocol: Arabidopsis seeds were surface-sterilized, plated on half-strength Murashige and Skoog media (without sucrose), and stratefied in darkness at 4 C for 5 days. After imbibition, the seeds were grown in 50 mmol m-2 s-1 red light at 21 C for 2 days (48 hours) to induce germination. Then, the seedlings were either kept at 21 C or transferred to 27 C under the same light condition for 2 addional days.  Samples were collected 96 h after imbibition.

extract_protocol:
Seedlings (50-100 mg) were collected by flash freezing in liquid nitrogen and stored at −80 °C until processing. Samples were ground to a fine powder in liquid nitrogen, and RNA was extracted using a Quick- RNA MiniPrep kit with on-column DNase I digestion (Zymo Research, Irvine, CA)
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers, followed by the second-strand cDNA synthesis.

value: whole seedling

tag: ecotype/background


title: RNA-Seq 35S:mim156, 27 C, rep1

description: 35S:mim156, 27 C, rep1


title: RNA-Seq 35S:mim156, 21 C, rep1

description: 35S:mim156, 21 C, rep1


title: RNA-Seq 35S:mim156, 21 C, rep3

description: 35S:mim156, 21 C, rep3


title: RNA-Seq 35S:mim156, 21 C, rep2

description: 35S:mim156, 21 C, rep2


title: RNA-Seq dcl1-24/hyl1-2, 27 C, rep3

description: dcl1-24/hyl1-2, 27 C, rep3


title: RNA-seq of wt stembryos at 120h, Time-course_rep1


title: RNA-Seq dcl1-24/hyl1-2, 27 C, rep2

description: dcl1-24/hyl1-2, 27 C, rep2


title: RNA-Seq dcl1-24/hyl1-2, 21 C, rep3

description: dcl1-24/hyl1-2, 21 C, rep3


title: RNA-Seq dcl1-24/hyl1-2, 27 C, rep1

description: dcl1-24/hyl1-2, 27 C, rep1


title: RNA-Seq dcl1-24/hyl1-2, 21 C, rep2

description: dcl1-24/hyl1-2, 21 C, rep2


title: RNA-Seq hyl1-2, 27 C, rep3

description: hyl1-2, 27 C, rep3


title: RNA-Seq dcl1-24/hyl1-2, 21 C, rep1

description: dcl1-24/hyl1-2, 21 C, rep1


title: RNA-Seq hyl1-2, 21 C, rep3

description: hyl1-2, 21 C, rep3


title: RNA-Seq hyl1-2, 27 C, rep1

description: hyl1-2, 27 C, rep1


title: RNA-Seq pif457, 27 C, rep3

description: pif457, 27 C, rep3


title: TCH3_D


title: TCH1_R


title: RNA-Seq pif457, 27 C, rep1

description: pif457, 27 C, rep1


title: RNA-Seq 35S:mim156, 27 C, rep3

description: 35S:mim156, 27 C, rep3


title: RNA-Seq hyl1-2, 21 C, rep1

description: hyl1-2, 21 C, rep1


title: RNA-Seq pif457, 27 C, rep2

description: pif457, 27 C, rep2


title: RNA-Seq hyl1-2, 21 C, rep2

description: hyl1-2, 21 C, rep2


title: RNA-Seq pif457, 21 C, rep2

description: pif457, 21 C, rep2


title: RNA-Seq Col-0, 27 C, rep1

description: Col-0, 27 C, rep1


title: RNA-Seq pif457, 21 C, rep1

description: pif457, 21 C, rep1


title: RNA-Seq Col-0, 21 C, rep3

description: Col-0, 21 C, rep3


title: RNA-Seq pif457, 21 C, rep3

description: pif457, 21 C, rep3


title: RNA-Seq Col-0, 21 C, rep2

description: Col-0, 21 C, rep2


title: RNA-Seq Col-0, 27 C, rep2

description: Col-0, 27 C, rep2


title: miRNA-Seq dcl1-24/hyl1-2, 27 C, rep2

description: dcl1-24/hyl1-2, 27 C, rep2

data_processing:
The sequencing data were analyzed using the homemade pipeline pRNASeqtools v0.8 (https://github.com/grubbybio/pRNASeqTools).
The raw reads were processed to remove the 3’ adapter sequence using cutadapt v3.0.
Trimmed reads of 18-42-nt were aligned to the Arabidopsis genome (Araport11) using ShortStack v3.899 with parameters.
Reads were normalized to 45S rRNA reads and RPMR (reads per million of 45S rRNA reads) values were calculated.
Differential miRNAs were identified by DESeq2 v1.35.0.
Assembly: Araport11
Supplementary files format and content: An Excel file includes miRNA abundance data (RPMR) for all samples.

extract_protocol:
Seedlings (50-100 mg) were collected by flash freezing in liquid nitrogen and stored at −80 °C until processing. Samples were ground to a fine powder in liquid nitrogen, and RNA was extracted using a Quick- RNA MiniPrep kit with on-column DNase I digestion (Zymo Research, Irvine, CA).
10 µg total RNA from 4-d-old seedlings grown under either 21oC or 27oC was resolved in a 15% urea-PAGE gel, and the 15-40-nt region was excised, and small RNAs were purified from the gel. Small RNA libraries were constructed using the NEBNext Multiplex Small-RNA Library Prep Set for Illumina (E7300).


title: miRNA-Seq dcl1-24/hyl1-2, 21 C, rep3

description: dcl1-24/hyl1-2, 21 C, rep3


title: miRNA-Seq dcl1-24/hyl1-2, 27 C, rep1

description: dcl1-24/hyl1-2, 27 C, rep1


title: RNA-Seq hyl1-2, 27 C, rep2

description: hyl1-2, 27 C, rep2


title: miRNA-Seq dcl1-24/hyl1-2, 21 C, rep2

description: dcl1-24/hyl1-2, 21 C, rep2


title: miRNA-Seq hyl1-2, 27 C, rep3

description: hyl1-2, 27 C, rep3


title: miRNA-Seq hyl1-2, 27 C, rep1

description: hyl1-2, 27 C, rep1


title: miRNA-Seq dcl1-24/hyl1-2, 21 C, rep1

description: dcl1-24/hyl1-2, 21 C, rep1


title: miRNA-Seq dcl1-24/hyl1-2, 27 C, rep3

description: dcl1-24/hyl1-2, 27 C, rep3


title: RNA-Seq Col-0, 21 C, rep1

description: Col-0, 21 C, rep1


title: miRNA-Seq hyl1-2, 27 C, rep2

description: hyl1-2, 27 C, rep2


title: miRNA-Seq hyl1-2, 21 C, rep2

description: hyl1-2, 21 C, rep2


title: miRNA-Seq hyl1-2, 21 C, rep1

description: hyl1-2, 21 C, rep1


title: miRNA-Seq Col-0, 27 C, rep2

description: Col-0, 27 C, rep2


title: miRNA-Seq Col-0, 27 C, rep3

description: Col-0, 27 C, rep3


title: RNA-Seq Col-0, 27 C, rep3

description: Col-0, 27 C, rep3


title: miRNA-Seq Col-0, 27 C, rep1

description: Col-0, 27 C, rep1


title: miRNA-Seq Col-0, 21 C, rep2

description: Col-0, 21 C, rep2


title: miRNA-Seq Col-0, 21 C, rep3

description: Col-0, 21 C, rep3


title: miRNA-Seq Col-0, 21 C, rep1

description: Col-0, 21 C, rep1


title: H9,rep1

status: Public on Feb 01 2023

city: Paris

first: Claire

last: Rougeulle

address: 35 rue Hélène Brion

country: France

institute: Université Paris Cité

data_processing:
Sequencing reads from both technical replicates were processed using the HiC-pro pipeline (Servant 2015), including, reads alignement on the human reference genome (hg38, mapping quality > 23) and valid interaction pairs detection. Raw contact matrices were built by binning in genomic windows of 2kb, 4kb, 6kb, 8kb and 10kb, using the allValidPairs2cooler.sh utility. Correlation analysis between the raw contact matrices of both replicates was done using the hicCorrelate package from the HiCExplorer toolbox (Wolff 2020). The 4kb binning was retained for the very good correlation score (R²=0,67) and the contact matrices of both replicates were merged. The raw contact interaction frequencies of the captured region were normalized using the Iterrative Correction approach (Imakaev 2012). Insulation score was computed on the normalized contact matrice using the hicFindTads package (Wolff 2020) with –minDepth 12000 –maxDepth 48000 –step 8000. Viewpoints data were extracted using the chicViewpoint package (Wolff 2020) with default parameters. Chromatin loops were called on ICE normalized contact matrices using the mustache software with default parameters (Ardakany 2020), non-significant loop interactions (FDR p.value < 0,05) were filtered before downstream analysis.
Assembly: hg38
Supplementary files format and content: Bigwig files for insulation score and viewpoints analysis
Supplementary files format and content: cool file containing the raw and the ICE normalized interaction frequency values binned at 4kb

source_name: Embryonic

growth_protocol: Primed hESC lines (H9 , obtained from the WiCell Research Institute, and WIBR2 , obtained from the Whitehead Institute for Biomedical Research) were cultured on Matrigel-coated culture dishes (BD Biosciences) in mTeSR™1 media (Stemcells technologies) according to the manufacturer instructions, in 20% O2 and 5% CO2 at 37°C. Primed hESCs were routinely passaged in clumps using a 0.5mM EDTA solution as previously described .

extract_protocol: In situ Capture HiC was performed as previously published (Davies 2016) with the following modifications. Cells were crosslinked with 2% formaldehyde. In short, 10x10⁶ cells were lysed on ice in 10 mM Tris-HCL pH8.0, 10 mM NaCl, 0,2% NP40, 1X complete protease inhibitor cocktail (Roche). De-crosslinking was performed in 100 µL 0,5% SDS at 37°C for 1h under agitation (1400 rpm). Nuclei permeabilization was done by adding 50 µL of 10% Triton X-100 and 290µL H2O and incubated for 15 minutes at 37°C and swirling at 1400 rpm. To prepare for digestion, 50µL of 10X DpnII buffer was added and 50µL were taken as an undigested control. At mid day, a first 10µL of DpnII (NEB, 50 000 U/ml) was added and incubated for 4 hours at 37°C, swirling at 1400 rpm. Another 10 µL was added and incubation was carried on overnight. A final 10µL of DpnII  was added the following morning and  left for 4 hours. DpnII was inactivated by heating at 65°C for 20 minutes and 50 µL were taken as an digested-unligated control. Ligation was carried by adding 800 µL of ligation cocktail containing 240U T4 ligase (ThermoScientific 30U/µL), 1X T4 ligase buffer, and incubated overnight at 16°C swirling at 1000 rpm. To prepare for DNA purification, samples and controls were incubated with 150µg of ProteinaseK (Eurobio) for 4 hours ar 65°C. Then, 0,1 vol. of NaAc 3M and 3 vol. of 100% EtOH were added and incubated overnight at -80°C. Precipitated DNA were pelleted by centrifugation, washed with 70% EtOH and finally air dryied. Samples and controls were resuspended in 100 µL and 20 µL H2O respectively and 1 µL of RNAse A (ThermoScientific) was added and incubated at 37°C for 1 hour.  DNA quantities in sample and controls were determined using the Qbit broad range kit (ThermoScientific) and 100 ng of each were loaded on an agarose gel as quality control of the digestion and ligation steps.

value: Embryonic

tag: cell line

value: Embryonic Stem Cell

value: Wild type


title: H9,rep2

tag: cell type


title: P90 wildtype harvested following 5h of sleep deprivation, replicate 7 [WTSD5_P90_PFC_7]

status: Public on Jan 16 2023

city: Spokane

first: Lucia

last: Peixoto

state: WA

address: PO Box 1495

department: College of Medicine

institute: Washington State University, Spokane

data_processing:
Raw sequencing reads in FASTQ files  were processed with Salmon v1.8 using the ‘mapping mode’. To prepare the index, Gencode (release M28) reference genome (‘GRCm39.primary_assembly.genome.fa.gz’) and reference transcriptome (‘gencode.vM28.transcripts.fa.gz)’ were downloaded, along with GTF coordinates (‘gencode.vM28.annotation.gtf.gz’). To improve the accuracy of quantification estimates from Salmon, an index was built that incorporated a set of genome targets as decoys. Using the concatenated list of transcriptome targets along with genome targets, the `salmon index` function was used to build the index with the flags: `--gencode`, `--threads 4`, and  `-k 31`.
`salmon quant` was used to perform quantification with the flags `--threads 6` and `--numBootstraps 30`. To create the final SummarizedExperiment object at the gene level, the summarizeToGene()` function from the tximeta (v1.14) R/Bioconductor package was used.
Assembly: GRCm39
Supplementary files format and content: text file, summarized experiment with gene counts (Tximeta) for gene level analysis

source_name: Pre Frontal Cortex

growth_protocol: Timed breeding pairs were established to obtain wild type (WT) at specific postnatal ages. Mice were house in standard cages on a 12:12 h light:dark cycle with food and water ad libitum. P16 mice were housed with the littermates and parents until the experiment. P24 mice were weaned and single housed at P18. P30 and P90 animals were weaned at P21 and single housed for a week before SD.

extract_protocol:
RNA was extracted from thawed tissue using the Qiagen RNAeasy Microarray Tissue kit with RWT buffer. The tissue was lysed and homogenized using a TissueLyzer. Briefly, 1ml of Qiazol was added to the pre-sterilized 2ml round bottom tubes with one 5mm steel bead inside,before adding the frozen tissue. The tissue was liyed for 2x 2min at 30Hz. Samples were spun down and the clear lysate was transfere to a clean tube. RNA was isolated through phase separation. For this, 200 µl of water saturated chloroform with isoamylalcohol (ChCl3:IAA 24:1) was added followed by vigorous shaking, incubated at rorom temparature of 3 min and then centrifuged at 12,000 g for 15 min at 4C. The aqueous phase was transfered to a new tube and 600ul of 100% EtOH was added. The ethanol-lysate mix was then loaded on a RNAeasy mini spin column and centrifuged at max speed for 30sec at room temparature. The loaded colums were washed using 500 µl of RWT (Qiagen), centrifuged again for 30s at max speed at room temparature. The flowthrough was discarded. The membrane was washed twice with 500 µl of RPE buffer (Qiagen) followed by a 30s (first wash) or 2min (second wash) spin at max speed at room temparature. RNA was eluted from the membrane with 30ul of RNase free water. The RNase free water was loaded directly onto the semi dry membran, centrifuged for one min at max speed at RT. This step was reapeated once by re-loading the eluat and centrigation again for 1min at room temparature. 1ul of the RNA eluat was used to measure concentrations in the Nanodrop. The extraced RNA sampel was aliquoted in 2x15ul aliquots and stored at -80C until further processing.
The integrity of total RNA was assessed using Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) with the High Sensitivity RNA Analysis Kit.  RNA Quality Numbers (RQNs) from 1 to 10 were assigned to each sample to indicate its integrity or quality.  RNA samples with RQNs ranging from 7 to 10 were used for RNA library preparation with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA).  Briefly, mRNA was isolated from 2.5 µg of total RNA using poly-T oligo attached to magnetic beads and then subjected to fragmentation, followed by cDNA synthesis, dA-tailing, adaptor ligation and PCR enrichment.  The sizes of RNA libraries were assessed by Fragment Analyzer with the High Sensitivity NGS Fragment Analysis Kit.  The concentrations of RNA libraries were measured by StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapabiosystems, Wilmington, MA).  The libraries were diluted to 2 nM with RSB (10 mM Tris-HCl, pH8.5) and denatured with 0.1 N NaOH.  Eighteen pM libraries were clustered in a high-output flow cell using HiSeq Cluster Kit v4 on a cBot (Illumina).  After cluster generation, the flow cell was loaded onto HiSeq 2500 for sequencing using HiSeq SBS kit v4 (Illumina).  DNA was sequenced from both ends (paired-end) with a read length of 100 bp.

treatment_protocol: Begining at light onset, sleep deprived mice were kept awake for 3 or 5 hours via gentle handling. P16 animals for SD or homecage control were separated from the dam and housed in groups of 2 or 3 in a prewarmed cage. Prefrontal cortex tissue was collected at the conclusion of sleep deprivation (SD) and immediately flash frozen in liquid nitrogen. Control animals that were left in their home cage to sleep were dissected at the same time of day (HC).

value: Pre Frontal Cortex

value: Brain Tissue Homogenate

value: wild type

value: Sleepdeprivation


title: P90 wildtype homecage control harvested at ZT hour 5, replicate 8 [WTHC5_P90_PFC_8]

value: control


title: P90 wildtype harvested following 5h of sleep deprivation, replicate 8 [WTSD5_P90_PFC_8]


title: P90 wildtype harvested following 5h of sleep deprivation, replicate 6 [WTSD5_P90_PFC_6]


title: P90 wildtype homecage control harvested at ZT hour 5, replicate 6 [WTHC5_P90_PFC_6]


title: P90 wildtype homecage control harvested at ZT hour 5, replicate 7 [WTHC5_P90_PFC_7]


title: P24 wildtype harvested following 3h of sleep deprivation, replicate 3 [WTSD3_P24_PFC_3]


title: P24 wildtype harvested following 3h of sleep deprivation, replicate 2 [WTSD3_P24_PFC_2]


title: P24 wildtype harvested following 3h of sleep deprivation, replicate 1 [WTSD3_P24_PFC_1]


title: P24 wildtype homecage control harvested at ZT hour 3, replicate 1 [WTHC3_P24_PFC_1]


title: gcgr-/- α cells

status: Public on Oct 26 2022

city: Shanghai

first: jihong

last: zheng

state: Shanghai

address: Siping Road, Yangpu District

country: China

institute: Tongji University

data_processing:
The tail ploy T of sequencing reads was first removed with customized scripts.
The adapter in reads was trimmed using Trim Galore (-q 25 --phred33 --length 10 --stringency 3).
Trimmed reads was aligned to the reads to the GRCz11 reference genome (UCSC) using STAR.
The reads in each cell was counted using featureCounts (Liao et al., 2014) (-T 5 -p -t CDS).
The gene expression matrix was generated by combining the expression of each cell. Finally, we got a matrix of 24900 genes and 576 cells (384 gcgr-/- α cells and 192 control α cells).
Genome_build: GRCz11
Supplementary_files_format_and_content: csv file were the final expression matrix of  576 cells (384 gcgr-/- α cells and 192 control α cells)

organism: Danio rerio

source_name: 7 dfp zebrafish larvae α cells_gcgr-/-

growth_protocol: Zebrafish (Danio rerio) were maintained in a recirculating aquaculture system with a 14h:10h darkness cycle at 28˚C.

extract_protocol:
7 dpf (days post fertilization) zebrafish larvae were sacrificed and digested gently with collagenase P (1.2mg/mL, Sigma-Aldrich) for 5 min at 37˚C, then the islets was picked out from the homogenate under fluorescent microscope (Lecia M205 FCA , Lecia Wetzlar, Germany) by tweezers manually, the isolated islets were digested again into single cell suspension with Liberase DH (100μg/mL, Roche) for 50 min at 36˚C. Finally, the GFP positive cells were collected by FACS (BD AriaIII) into 96 well plate containing lysis buffer.
Single-cell 3’ end libraries was generated using modified Smart-seq2 protocol.

value: gcgr-/-

value: islets

value: alpha cells

value: 7 dfp


title: P24 wildtype homecage control harvested at ZT hour 3, replicate 2 [WTHC3_P24_PFC_2]


title: miRNA-Seq hyl1-2, 21 C, rep3

description: hyl1-2, 21 C, rep3


title: Cell-4 [20E+Ecoli+ML]

status: Public on Oct 04 2022

city: St Petersburg

first: Rui

last: Zhou

state: FL

address: REB 4130, 501 6th Ave S

institute: Johns Hopkins University

description: S2 cells first treated with 1 μM 20-hydroxyecdysone for 24 hours and subsequently with a mixture of E. coli 1106 and M. luteus for an additonal 48 hours

data_processing:
Illumina’s bcl2fastq was used to convert bcl files to FASTQ files and de-multiplex the samples.
The raw reads were mapped to reference genome (dm3) using bowtie2.
The find_circ v1.0 program was applied to identify circRNAs using the unmapped reads.
Genome_build: dm3
Supplementary_files_format_and_content: circRNAs bed file for each sample

organism: Drosophila melanogaster

source_name: cultured S2 cells

growth_protocol: Drosophila S2 cells are maintained in Schneider’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen).

extract_protocol:
Total RNA was extracted using TRIzol reagent (Invitrogen).
Total RNA samples were first subjected to ribosomal RNA depletion using Ribo-zero rRNA removal kit, and subsequently treated with RNase R for 30 minutes at 37oC.  The resulting enriched circular RNA fraction were subjected to cDNA library construction using illumine kit.

treatment_protocol: Drosophila S2 cells (~ 2X10^6 cells/mL) were 1) left untreated, 2) treated with 1 M 20-hydroxyecdysone for 24 hours, 3) treated with a mixture of overnight cultures of E. coli 1106 and M. luteus for 48 hours, or 4) treated with 1 M 20-hydroxyecdysone for 24 hours and subsequently with a mixture of overnight cultures of E. coli 1106 and M. luteus for an additional 48 hours.

value: embryo-derived S2 cells

tag: passages

value: passages 10-15

value: first treated with 1 uM 20-hydroxyecdysone for 24 hours and subsequently with a mixture of E. coli 1106 and M. luteus for an additonal 48 hours


title: P24 wildtype homecage control harvested at ZT hour 3, replicate 3 [WTHC3_P24_PFC_3]


title: Cell-2 [20E]

description: S2 cells treated with 1 μM 20-hydroxyecdysone for 24 hours

value: treated with 1 uM 20-hydroxyecdysone for 24 hours


title: Cell-3 [Ecoli+ML]

description: S2 cells treated with a mixture of E. coli 1106 and M. luteus for 48 hours


title: Elav-sh3

data_processing:
Illumina’s bcl2fastq was used to convert bcl files to FASTQ files and de-multiplex the samples.
The raw reads were assessed with FastQC v0.11, then mapped to dm6 genome using HISAT2 aligner (version 2.0.4) with default parameters.
Partek Genomics Suite (version v7.17.0918) was applied for gene quantification based on UCSC RefSeq annotation (dm6. refFlat.txt.gz, v28-May-2017).
Genome_build: dm6
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: tab-delimited text file include RPKM values for every gene and every sample

source_name: fruitfly heads

growth_protocol: Fly stocks are maintained on a standard fly food (Nutri-Fly, molasses formulation) and kept at 25 oC.

extract_protocol:
Total RNA was extracted using TRIzol reagent (Invitrogen).
Total RNA samples were first subjected to ribosomal RNA depletion using Ribo-zero rRNA removal kit. The resulting samples were subjected to cDNA library construction using illumine kit.

treatment_protocol: We crosses flies carrying the UAS-shEdis-A or UAS-shGFP (control) transgenes to the pan-neuronal Gal4 driver line, Elav-Gal4. Groups of male progeny (3-5 d of age, ~50 flies per group) of the indicated genotypes were collected and total RNA samples were extracted from fly heads. Two biological replicates of control samples (Elav>shGFP1 and Elav>shGFP3) and three biological replicates of Edis knockdown samples (Elav>shEdis2, Elav>shEdis3 and Elav>shEdis23) were employed in this study.

tag: gender

value: male

value: Elav>shEdis-A

value: fruitfly heads

tag: strain background

value: progeny of crosses between Elav-Gal4 and UAS-shEdis-A (at attP2)


title: Elav-GFP1

tag: genotype

value: Elav>shGFP

value: progeny of crosses between Elav-Gal4 and UAS-shGFP (at attP2)


title: Elav-sh23


title: Elav-sh2


title: ChIP_KMS27_GAPMER_NEAT1_2

status: Public on Jul 06 2022

city: Rome

first: Maurizio

last: Fanciulli

address: Via Elio Chianesi 53

department: Department of Research, Advanced Diagnostic, and Technological Innovation

country: Italy

institute: IRCCS Regina Elena National Cancer Institute

data_processing:
Sequence reads were mapped to hg19 by using bowtie2 v.2.3.5.1 with parameters -p6 -t --local.
Peaks abundance of RNA-seq was computed by using kallisto quant with parameter -b 100 and transcriptome index of hg19.
Differential analysis was executed thourgh Sleuth R package by computing full and reduced models.
Peaks calls were obtained by utilizing macs2 callpeaks with parameters --broad -B -q 0.1 -g hs; for DRIP-seq data also the parameter -c has been used.
The macs peaks where matched against a customized master list of TSS regions and their abdundance is quantified through bedtools multicov tool.
The bedgraph outputs from macs were converted into bigwig file by using bedGraphToBigWig tool.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig file;

source_name: bone marrow

extract_protocol:
For Rna-seq total RNA was extracted from the same number of viable cells using QIAzol Lysis Reagent (Qiagen), purified from DNA contamination through a RNase-Free DNase Set (Qiagen) digestion step and further enriched by Qiagen miRNeasy mini Kit (Qiagen)
ChIP seq : lysates were clarified from sonicated nuclei and DNA complexes were isolated with antibody. DRIP-seq: Genomic DNA was extracted in according with the manual of Blood & Cell Culture DNA Kits (n° cat 13323) and then subjected to  immunoprecipitation by S9.6 antibody.
For ChIP seq libraries were prepared in according with ChIPmentatio protocol (Christian Schmidl et al , nature methods 2015) using TN5 transposase. For DRIP seq Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
ChIRP seq was performed using H.Y. Chang (JOVE 2012) protocol and the library was constructed usign Acel-NGS 2S plus DNA library kit (Swift)

value: gapmer NEAT1


title: Elav-GFP3


title: ChIP_KMS27_GAPMER_NEAT1_1


title: ChIP_KMS27_Control_1


title: DRIP_KMS27_siChe

description: sorted_siChe-DrIP.fastq.gz_treat_pileup.bw

data_processing:
Library strategy: DRIP-seq
Sequence reads were mapped to hg19 by using bowtie2 v.2.3.5.1 with parameters -p6 -t --local.
Peaks abundance of RNA-seq was computed by using kallisto quant with parameter -b 100 and transcriptome index of hg19.
Differential analysis was executed thourgh Sleuth R package by computing full and reduced models.
Peaks calls were obtained by utilizing macs2 callpeaks with parameters --broad -B -q 0.1 -g hs; for DRIP-seq data also the parameter -c has been used.
The macs peaks where matched against a customized master list of TSS regions and their abdundance is quantified through bedtools multicov tool.
The bedgraph outputs from macs were converted into bigwig file by using bedGraphToBigWig tool.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig file;

value: Che1 interference


title: DRIP_Rnase_Control

value: control


title: DRIP_Rnase_siChe


title: DRIP_KMS27_Control


title: ChIRP_KMS27_Control


title: control  α cells

source_name: 7 dfp zebrafish larvae α cells_control

value: wild type


title: ChIRP_KMS27_siChe


title: RNA_KMS27_siChe_3

description:
Table_differ_analysis_sleuth_wald_test_RNA_seq_siChe.txt
Table_reads_count_normalized_in_tpm_RNA_seq_siChe.txt


title: RNA_KMS27_Control_1


title: RNA_KMS27_siChe_1


title: RNA_KMS27_GAPMER_NEAT1_1

description:
Table_differ_analysis_sleuth_wald_test_RNA_seq_gapmerNEAT1.txt
Table_reads_count_normalized_in_tpm_RNA_seq_gapmerNEAT1.txt


title: RNA_KMS27_Control_2


title: RNA_KMS27_GAPMER_NEAT1_2


title: RNA_KMS27_siChe_2


title: RNA_KMS27_GAPMER_NEAT1_3


title: RNA_KMS27_Control3


title: RNA_KMS27_Control_3


title: RNA_KMS27_Control1


title: ChIP_KMS27_Control_2


title: H1299 with glucose and NH4Cl rep2

status: Public on Apr 30 2022

city: Ann Arbor

first: Yue

last: Zhao

address: 100 Washtenaw Avenue

department: Computational Medicine and Bioinformatics

institute: University of Michigan

data_processing:
Sequenced reads were mapped to human reference genome (GRCh38p12) via HISAT2 v2.1.0
The raw read counts on genes were derived by featureCounts from the subread package v1.5.1 in unstranded mode, based on Ensembl gene annotation release 92.
Assembly: GRCh38p12
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: human non-small cell lung carcinoma cells

growth_protocol: H1299 cells (100X10^4 cells/10 cm dish) were cultured in full RPMI 1640 medium supplemented with 5% FBS for 24 hr.

extract_protocol:
Total RNA from treated H1299 cells was extracted using the Total RNA Purification Plus kit (#48300, NORGEN BIOTEK CORP., Canada), followed by quality assessment by NanoDrop One (#70-105-8111, Thermo Fisher Scientific, USA).
For mRNA library generation, 200 ng of total RNA was treated with NEBNext Poly mRNA Magnetic Isolation Module (#E7490L, New England Biolabs, USA) following the manufacturer’s protocol. Subsequently, isolated mRNA was fragmented for 10 min. cDNA was synthesized and amplified for 12 PCR cycles using NEBNext Ultra II Directional (stranded) RNA Library Prep Kit for Illumina (#E7760L, New England Biolabs, USA) with NEBNext Multilex Oligos Indexes kit following the manufacturer’s directions.

treatment_protocol: Triplicate cells were then washed with PBS once and treated in fresh serum-free RPMI 1640 medium with or without glutamine (4 mM) or glucose (5 mM) or NH4Cl (4 mM) for 12 hr.

value: d lymph node

tag: tumor type

value: human non-small cell lung carcinoma

value: treated in fresh serum-free RPMI 1640 medium with glucose (5 mM) and NH4Cl (4 mM)


title: H1299 with glucose and NH4Cl rep3

value: human non-small cell lung carcinoma cells

tag: treatment


title: Cell-1 [S2]

description: non-treated S2 cells

value: embryo-derived S2 cells

value: passages 10-15

tag: strain

value: non-treated


title: H1299 with glucose and NH4Cl rep1

tag: tissue


title: H1299 with glucose and glutamine rep2

value: treated in fresh serum-free RPMI 1640 medium with glutamine (4 mM) and glucose (5 mM)


title: H1299 with glucose and glutamine rep1


title: H1299 with glutamine alone rep3

value: treated in fresh serum-free RPMI 1640 medium with glutamine (4 mM) alone


title: H1299 with glutamine alone rep2


title: H1299 with glucose alone rep2

value: treated in fresh serum-free RPMI 1640 medium with glucose (5 mM) alone


title: H1299 no glucose no glutamine rep3

value: treated in fresh serum-free RPMI 1640 medium without glutamine and glucose


title: H1299 with glucose alone rep3


title: H1299 with glutamine alone rep1


title: RNA_KMS27_Control2


title: H1299 with glucose alone rep1


title: T-Short Frozen 6c

status: Public on Mar 15 2022

city: Philadelphia

first: Eoin

middle: Christopher

last: Whelan

state: PA

address: 3800 Spruce St

department: Biomedical Sciences

institute: University of Pennsylvania School of Veterinary Medicine

data_processing:
Fastq files generated with BaseSpace (Illumina)
Count matricies generated with indrops.py (github.com/indrops) using the rat and mouse genome builds listed below using Bowtie v1.2.3. These are the processed files listed above (i.e. tsv files)
QC, filtering and UMAP generated with Seurat v.3.1
Pseudotime generated with Monocle v3
Pathway analysis using Ingenuity Pathway Analysis (Qiagen)
Genome_build: Rattus norvegicus 6.0 DNA primary assembly (Rattus_norvegicus.Rnor_6.0.dna_sm.primary_assembly.fa.gz) along with the corresponding GTF file (Rattus_norvegicus.Rnor_6.0.85.gtf.gz) from Ensembl along with the Mus_musculus.GRCm38.dna_sm.primary_assembly.fa and Mus_musculus.GRCm38.85.gtf.gz GTF files for mouse.
Supplementary_files_format_and_content: csv file containing contrasts between each treatment and the 0ng FGF control

organism: Rattus rattus

source_name: testis

growth_protocol: Fresh tissue from rat testes were used as appropriate. Cells were digested in collagenase, trypsin and Miltenyi Dead Cell Removal Kit before encapsulation. For transplantation experiments, EpCAM+ cells were used to transplant into J:Nu nude mice and allowed to grow for 2+ months before being harvested in the same manner.

extract_protocol:
Cells were encapulated in a salinized chip and RT reaction was performed at 70°C instead of 65°C but otherwise followed the inDrops protocol.
Library generated per inDrops protocol. Sequenced on NextSeq500 (Illumina) 75 cycle-cycle high output to depth of approximately 30k reads per cell

treatment_protocol: Cells for transplantation or encapsulation were either used fresh, frozen for 1-4 months or frozen for 23+ years. Cells were used unselected or following EpCAM MACS antibody selection.

tag: replicate

value: Transplanted Short Frozen

tag: selection

value: Unselected


title: H1299 no glucose no glutamine rep1


title: T-Short Frozen 6b


title: H1299 no glucose no glutamine rep2


title: T-Short Frozen 5a


title: T-Short Frozen 5b


title: T-Short Frozen 4a


title: H1299 with glucose and glutamine rep3


title: T-Short Frozen 3a


title: T-Short Frozen 2a


title: T-Long Frozen 2c

value: Transplanted Long Frozen


title: T-Short Frozen 2b


title: T-Long Frozen 2b


title: T-Long Frozen 4b


title: T-Long Frozen 2a


title: T-Long Frozen 4c


title: T-Long Frozen 3b


title: T-Long Frozen 1a


title: T-Fresh5b

value: Transplanted Fresh


title: T-Fresh 2a


title: Fresh 9c

value: Fresh


title: Fresh 8b


title: Fresh 7a


title: T-Short Frozen 6a


title: Fresh 5b


title: T-Fresh 3a


title: T-Fresh4b


title: T-Short Frozen 2c

value: EpCAM+


title: T-Short Frozen 3c


title: Fresh 5a


title: T-Long Frozen 4f


title: T-Short Frozen 5c


title: T-Long Frozen 4e


title: T-Long Frozen 3f


title: T-Long Frozen 2e


title: T-Long Frozen 1d


title: T-Fresh 5c


title: T-Fresh 4c


title: T-Fresh 1c


title: T-Fresh 3d


title: Long Frozen 3b

value: Long Frozen


title: Short Frozen 3b

value: Short Frozen


title: Long Frozen 3a


title: Long Frozen 2a


title: Fresh 8c


title: Fresh 7c


title: Fresh 6d


title: Fresh 7d


title: Fresh 6c


title: T-Long Frozen 3d


title: Fresh 5c


title: NlaIII5plusplusnew2 : Stable population sample B2

status: Public on Nov 22 2021

city: Rotterdam

first: Tokameh

last: Mahmoudi

address: Dr Molewaterplein 40

country: Netherlands

institute: Erasmus Medical Center

data_processing:
Base calling was performed using software provided with the Ion Torrent Proton sequencer.
The FASTQ files were processed to remove gene trap vector primers. To this end, we used R/Bioconductor packages ShortRead and Biostrings to match primer sequences within the sequenced reads, allowing for two mismatches at maximum for each primer sequence. The identified primers were trimmed and reads shorter than 15 bp were discarded. The remaining reads were aligned to the human reference genome version hg19 retrieved from the Illumina iGenomes repository, using bwa mem.
The produced BAM files were subjected to integration site identification. To this end, we re-implemented the HaSAPPy algorithm in R. We added attributes for i) filtering out read artifacts from introns, based on the overall intronic read distribution for each sample, ii) selecting the fraction of neighboring (Independent Insertions, Disrupting Insertions, Bias) triplets for LOF analysis and iii) library normalization across samples. After the quality control steps, our HaSAPPY reimplementation assigns a Local Outlier Factor (LOF) score to each gene in a sample based on a triplet score derived from the number of putative GT integrations in the sample compared to the reference, additionally taking into account the potential bias arising from unbalanced sense and anti-sense read numbers.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited files containing HaSAPPy triplet read counts.

source_name: myeloid leukemia

growth_protocol: KBM7 (a kind gift from Thijn Brummelkamp) and Hap-Lat (latently HIV-1-infected KBM7-derived cell line) cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (ThermoFisher Scientific) supplemented with 10% fetal calf serum (FCS) and 2% penicillin-streptomycin (Pen/Strep). Haploidy of KBM7 cells was maintained by periodically sorting cells for size (5% smallest). Ploidy of cells was determined by propidium iodide staining using Jurkat cells as a diploid control.

extract_protocol:
Genomic DNA was isolated from 5 million cells using the DNeasy kit (Qiagen). Four micrograms of genomic DNA (gDNA) was digested with NlaIII or MseI. After PCR spin column purification (Qiagen), 50 ml of eluted digested DNA was ligated using T4 DNA ligase (Roche) in a volume of 2 ml. The reaction mix was purified using spin columns and used as the template in a PCR with primers annealing to internal sequences in the gene-trap vector (59-CTGCAGCATCGTTCTGTGTT-39 and 59-TCTCCAAATCTCGGTGGAAC-39). The resulting PCR products were used for library preparation.
Sequencing libraries were created using the Ion Plus Fragment Library Kit (ThermoFisher Scientific) according to manufacturer’s instructions, with minor modifications: briefly, 15 ng of the PCR products were diluted with ddH2O to a final volume of 39 μl. The samples were end repaired, adaptor ligated and amplified; this was followed by 2 rounds of purification using Agencourt AMPure XP beads. Library qualities and quantities were assessed by Bioanalyzer, using the DNA High Sensitivity Kit (Agilent Technologies). The quantified libraries were pooled together in 10plex, at a final concentration of 40 pM. Templating, enrichment and chip loading was performed on an Ion Proton Chef system using the Ion PI™ Hi-Q™ Chef Kits (ThermoFisher Scientific); sequencing was performed on an Ion Proton PI™ V3 chip, with the Ion PI™ Hi-Q™ Sequencing 200 Kit, on an Ion Proton instrument (ThermoFisher Scientific), according to manufacturer’s instructions.

treatment_protocol: Minimal HIV-1 (LTR-GFP, HIV-1-658) virus was produced by transfection of 293T cells in 15-cm culture dishes using polyethylenimine (PEI) (Sigma-Aldrich) with a mixture of 6.8 mg p658, 2 mg vesicular stomatitis virus G (VSVG), and 4.5 mg Gag-Pol plasmids. Haploid KBM7 cells were infected with HIV-1-derived virus at low MOI such that approximately 5 to 10% of cells became productively infected as determined by GFP expression. Five days after infection, the GFP-negative cell population harboring uninfected and potentially latently infected cells was sorted by flow cytometry-activated cell sorting (FACS) and stimulated with 350 nM suberoylanilide hydroxamic acid (SAHA) (Selleck Chem), and 5 ng/ml TNF-a (Sigma-Aldrich). Twenty-four hours poststimulation, GFP-positive cells were single cell-sorted by FACS into 96-well plates. Clones were expanded and characterized for their basal GFP expression and their potential for reactivation. From the clonal cell lines generated, Hap-Lat was selected for low background and relative high reactivation upon stimulation (GFP-negative under basal conditions but amenable to induction to express GFP upon activation). Gene-trap virus was produced by transfection of HEK293FT cells in 15-cm culture dishes using 180 ml polyethylenimine (PEI) with a mixture of 8 mg pGT-mCherry, 2.1 mg pAdvantage, 3.1 mg cytomegalovirus (CMV)-VSVG, and 4.8 mg Gag-Pol plasmids in at total volume of 1 ml serum-free RPMI medium. After 12 h the growth medium was changed to fresh FCS and G418-supplemented RPMI. The virus was collected at 12, 24, 36, and 48 h. For the GT mutagenesis, 192 million Hap-Lat cells were preincubated with 100 mM deoxynucleoside triphosphates (dNTPs) (Invitrogen) 1 h before transduction. Cells were resuspended in 192 ml of undiluted GT virus. Spin infection was performed for 90 min at 1,500 rpm in 24-well plates containing 2 million cells per well and 8 ml protamine sulfate per ml. After 24 h cells were subjected to a second round of infection after which virus was removed, and cells were left to recover in supplemented IMDM. We estimate that this sequential infection resulted in 1 to 4 GT integrations per cell, with the majority of cells containing one integration. A first round of sorting was performed 8 days after the second infection. Subsequently, between 500,000 and 1 million GFP-mCherry double-positive cells were sorted each day for 5 consecutive days using two cell sorters (BD FACSAria II SORP and BD FACSAria III). Sorted cells were pooled, left to recover, and expanded for the next round of sorting. A second round of sorting was performed 17 days after the second infection. Between 2.6 and 3 million cells were collected each day for 3 consecutive days using two sorters. These cells were pooled and left to recover and expand for a third round of sorting on day 22 after the second infection, yielding 6.1 million cells from 1-day sorting on two machines. On day 29 after the second infection a fourth round of sorting was performed, yielding 7.2 million cells from 1-day sorting on two machines. After the cells of the fourth round of sorting were left to recover and expand, a subpopulation of highly mCherry and GFP double-positive cells were apparent. In a final fifth round of sorting the total GFP-positive, mCherry-positive population (10.1 million cells) was sorted as soon as enough cells were available (day 33 after the second infection) while the GFP-mCherry-positive stable subpopulation (5.9 million cells) was cultured for several more days and sorted (>>35 days after the second infection). After recovery, genomic DNA was isolated from the sorted cell populations, as well as from a population of mutagenized unsorted cells.

value: myeloid leukemia

value: genetrap


title: Latent HIV-1 infected Jurkat cells, Input chromatin

status: Public on Feb 06 2022

data_processing:
Base calling was performed using software provided with the Ion Torrent Proton sequencer.
In order to align the short reads produced by the ChIP-Seq procedure and also capture reads mapped to the HIV genome, we constructed a custom version of the human genome (UCSC version hg38) where we attached the HIV genome as an additional chromosome. We used the HIV strain K03455.1. Subsequently, the resulting genome was indexed and reads were aligned with bwa mem 0.7.17. In order to compensate for differences in library sizes between ChIP and Input DNA sequencing samples, the total number of reads of each sample was equalized by uniformly downsampling reads relatively to the sample with the lower number of reads.
ChIP peaks were then called with MACS2 using default parameters. The peaks returned by MACS2 were further filtered by i) imposing an additional MACS FDR threshold of 0.1%, ii) excluding peaks demonstrating a fold enrichment less than 3 in log2 scale (where fold enrichment is the ratio of normalized reads under a peak area in the ChIP sample to the respective number of normalized reads in the Input DNA sample).
Genome_build: hg38
Supplementary_files_format_and_content: BigWig signal files for ChIP-Seq data.

source_name: T-cell lymphoma cell line

growth_protocol: Latent HIV-1 infected Jurkat cells (clone J-Lat 11.1 stably expressing HIV-LTR-HS2-guided HA-V5-FLAG-dCas9) were cultured in RPMI-1640 media supplemented with 7% FBS and 100 µg/ml penicillin-streptomycin at 37°C in a humidified 95% air, 5% CO2 incubator.

extract_protocol:
ChIP-sequencing was performed using the sequentially purified chromatin, first immunoprecipitated using V5 affinity beads, eluted and re-immunoprecipitated using Histone H3-H2B affinity purification. Approximately 3 billion cells per condition were collected, cross-linked and processed in batches of 400 million cells. After collection, by centrifugation at 800 rcf for 5 minutes, each batch was washed two times in 40 ml PBS supplemented with 1 mM MgCl2 and 1 mM CaCl2, and cross-linked in 40 ml PBS with 1% formaldehyde (Formaldehyde methanol free 16%, Polysciences inc.) at room temperature for 30 min with vertical rotation at 15 rpm. The reaction was then quenched with the addition of 1 M glycine to a final concentration of 125 mM. Cross-linked cells pellet were then washed in 20 ml cold buffer B (0.25% Triton x-100, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6) and 20 ml cold buffer C (0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES pH 7.6), resuspended in 2 ml ChIP incubation buffer (1% Triton x-100, 150 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, 20 mM HEPES pH 7.6, protease inhibitor cocktail tablets (EDTA-free, Roche)) and then transferred to 15 ml polystyrene Falcon tubes compatible with sonication. In order to isolate and expose the cross-linked nuclei to the subsequent denaturation steps the samples were sonicated with 15 ml probes at medium intensity for 10 minutes (30’ ON/30’ OFF intervals; Diogenode Bioruptor) and the cell nuclei were spun at 800 rcf for 10 minutes. The pellets were then re-suspended in 2 ml ChIP incubation buffer containing 0.15% SDS and sonicated again for 10 minutes at medium intensity (30’ ON/30’ OFF intervals; Diogenode Bioruptor). The sonicated nuclei in suspension were then aliquoted (500 µl per tube) in 2 ml Eppendorf tubes and denatured for 5 minutes at room temperature with 350 µl 10% SDS and 1.2 ml 8M Urea buffer (10 mM Tris pH 7.4, 1 mM EDTA and 8 M urea). The insoluble fraction of the nuclei, enriched for chromatin, was then precipitated by centrifugation at 16200 rcf at room temperature for 20 minutes. The precipitated pellets were then resuspended and combined (4 into 1) in a single 2 ml Falcon tube and denatured again for 5 minutes at room temperature with 500 µl of 4% SDS buffer (50 mM Tris ph 7.4, 10 mM EDTA, 4% SDS) and 8 M Urea buffer and then precipitated again by centrifugation at 16200 rcf at room temperature for 20 minutes.   The pellets were then resuspended and washed by centrifugation at 16200rcf at room temperature for 20minutes in 2ml 4% SDS buffer and then resuspended in 2.5 ml ChIP incubation buffer containing 1% SDS. The content of each tube was then transferred in a 15ml polystyrene tube and sonicated (3 tubes at the time) at a high intensity (30’ ON/30’ OFF intervals; Diogenode Bioruptor), using metal probes, to obtain chromatin fragments in a 200-500bp size range. The different chromatin batches were then pulled together concentrated through 100kDa Centricon filters, to buffer exchange with 0.1% SDS ChIP incubation buffer and remove smaller proteins and fragments. The sonicated chromatin (a total of 50 ml per condition) was then spun at max speed (20817 rcf) at 4°C for 30 minutes to remove cellular debris and diluted to 50 ml per condition. The chromatin was then precleared overnight at 4°C with vertical rotation at 15rpm (a total of 5 reactions per condition in 15ml tubes) with 500µls of a mix of Protein G and A SepharoseTM 4 Fast flow (GE Healthcare) beads and then used for immunoprecipitation. dCas9 containing complexes were isolated from the sonicated chromatin using 500µls (a total of 5 reaction where 100µls of affinity beads were used to purify 10ml of chromatin) of Anti-V5 Agarose Affinity Gel (Sigma) and then washed two times per each buffer (5minutes, 15rpm vertical rotation washes) with buffer 1 (0.1% SDS, 0.1% DOC, 1% Triton x-100, 150mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA, 20mM HEPES pH8.0.), buffer 2 (500mM NaCl: 0.1% SDS, 0.1% DOC, 1% Triton x-100, 500mM NaCl, 1 mM EDTA pH 8.0, 0.5mM EGTA, 20mM HEPES pH8.0), buffer 3 (0.25M LiCL, 0.5%DOC, 0.5% NP-40, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 20mM HEPES pH 8.0), buffer 4 (1mM EDTA pH8.0, 0.5mM EGTA pH 8.0, 20mM HEPES pH 8.0) to remove unspecific binding and eluted in a total volume of 1 ml elution buffer 1% SDS, 0.1M NaHCO3 per each condition. Dna was purified by phenol-chloroform extraction and EtOH precipitation.
DNA libraries were created using the Ion Plus Fragment Library Kit and protocol (c/n: 4471252, MAN0009847, ThermoFisher Scientific) with the follwoing modifications: the fragmented DNA was end-repaired and, after 30 min incubation at RT, was purified with Agencourt AMPure XP beads (Beckman Coulter). Adaptors were ligated and assigned BCs were added to the mixture followed by 30 min incubation at RT and a second purification. The mixture was amplified for 17 cycles and purified once more using a 2-step purification protocol. The eluted samples were quality-controlled and quantified on a bioanalyzer, according to manufacturer's instructions.

value: T-cell lymphoma

tag: fraction

value: Input


title: Fresh 5d


title: NlaIII5old: Total population sample D1


title: NlaIII5plusplusnew: Stable population sample B1


title: Latent HIV-1 infected Jurkat cells, immunoprecipitated chromatin

value: IP


title: NlaIII5old2: Total population sample D2


title: Short Frozen 4a


title: NlaIII5plus: Total population sample C


title: T-Fresh 2e


title: T-Fresh 2d


title: Long Frozen 4a


title: Short Frozen 2a


title: NlaIII5plusplus: Stable population sample A


title: MseIRef: Input Mse1


title: NlaIIIref: Input NlaIII


title: Fresh 9d


title: 07+08-Limbus pool

status: Public on Oct 25 2021

city: Maastricht

first: Vanessa

last: LaPointe

address: Universiteitssingel 40

institute: MERLN Institute

data_processing:
BCL files resulting from sequencing were transformed to FASTQ files with 10x Genomics Cell Ranger mkfastq.
FASTQ files were mapped with Cell Ranger count. During sequencing, 28 bp read 1 were used for identification of the Illumina library barcode, cell barcode and UMI. Read 2 was used to map to the reference transcriptome GRCh38. Filtering of empty barcodes was done in Cell Ranger.
The data from all samples were loaded in R (version 3.6.2) and processed using the Seurat package (version 3.2.0). More specifically, cells with at least 1000 UMIs per cell and less than 20% mitochondrial gene content were retained for analysis. The data of all 10x libraries was merged and processed together. The merged dataset was normalized for sequencing depth per cell and log-transformed using a scaling factor of 10,000. The patient effect was corrected using the integration function of Seurat and used for dimensionality reduction and clustering of all cells or cells selected per layer. Cells were clustered using graph-based clustering and the original Louvain algorithm was utilized for modularity optimization.
Genome_build: Human: Hg38
Supplementary_files_format_and_content: filtered_counts.csv; final dataset containing the expression counts for all genes across filtered cells
Supplementary_files_format_and_content: metadata_percell.csv; file containing metadata per single cell
Supplementary_files_format_and_content: matrix.mtx.gz; barcodes.tsv.gz, features.tsv.gz; cell ranger output for each 10X library

source_name: Cornea

growth_protocol: Both male and female donor corneas, with ages ranging from 22 to 79 years and preserved either in Optisol-GS (Corneas 01, 02, 03 and 04) at 4°C or in Organ culture media (Corneas 05, 06, 07 and 08) at 31°C were used.  Organ culture media comprised the following: minimum essential medium supplemented with 20 mM HEPES, 26 mM sodium bicarbonate, 2% (v/v) newborn calf serum (Thermo Fisher Scientific), 10 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin.

extract_protocol:
Manual dissection of the corneal tissue: Eight donor corneas were dissected for scRNAseq. First, cells were isolated from six corneas that were manually dissected to separate the epithelial, stromal and the endothelial layers to ensure the gentlest possible enzymatic dissociation of each layer. The corneas were vacuum fixed in a punch base (e.janach) endothelial-cell side up, stained with trypan blue solution (0.4%) for 30 s and washed with BSS ophthalmic irrigation solution. The corneal endothelium was then gently lifted following the Schwalbe line using a DMEK cleavage hook (e.janach) and fully stripped using angled McPherson tying forceps. The remaining tissue was trephined using a 9.5 mm Ø Barron vacuum punch (Katena) in order to separate the epithelium and stroma from the scleral ring. The remaining two corneas were used for limbus isolation. A surgical scalpel was used to cut the limbus into approximately 1 × 2 mm fragments, which were then rinsed with PBS.    Tissue dissociation to single cells: The six manually dissected corneal tissues were enzymatically treated to obtain single cell suspensions. The stripped corneal endothelium was incubated with 2 mg/mL collagenase (Sigma) solution in human endothelial serum free media (SFM) (Thermo Fisher Scientific) for 1–2 h at 37°C followed by a 10 min incubation with Accutase (Thermo Fisher Scientific) at 37°C to obtain a single cell suspension. The cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of human endothelial SFM media.    The corneal epithelium–stroma tissues were treated with 1.5 mg/mL collagenase and 0.2 mg/mL bovine testis hyaluronidase (Sigma) in DMEM/F12 (Thermo Fisher Scientific) for approximately 1 h at 37 °C. The released corneal epithelium was suctioned with a P1000 micropipette and further treated with Accutase for 10 min at 37°C to obtain a single cell suspension of corneal epithelial cells. The cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of DMEM/F12. The remaining corneal stroma was treated with 1.5 mg/mL collagenase and 0.2 mg/mL bovine testis hyaluronidase in DMEM/F12 for 5–7 h to obtain a single cell suspension of corneal keratocytes. After the incubation, the cells were centrifuged for 5 min at 800 × g and resuspended in 0.5 mL of DMEM/F12 media.    The limbus fragments isolated from the two corneas for limbal isolation were digested with four trypsinization cycles. In each cycle, the limbus fragments were incubated in 10 mL of 0.05% trypsin/0.01% EDTA (Thermo Fisher Scientific) at 37°C for 30 min. The trypsin containing dissociated cells were transferred to a 50 mL centrifuge tube containing 10 mL DMEM/F12 media and centrifuged for 5 min at 300 × g, after which the cells were resuspended in 0.5 mL of DMEM/F12 media. The undigested limbal fragments were placed again in 10 mL of 0.05% trypsin/0.01% EDTA for the following trypsinization cycle. The cells isolated from the limbus of the two donor corneas were pooled for the following steps.    Methanol cell fixation  After dissociation, the single cell suspensions were passed through a 100 μm Ø cell strainer, centrifuged for 5 min at 300 × g and resuspended in 1 mL ice-cold PBS to eliminate any medium remnants. Next, the cells were again centrifuged for 5 min at 300 × g and resuspended in ice-cold PBS at a ratio of 200 μL PBS per 1×10^6 cells followed by the dropwise addition of ice-cold methanol, at a ratio of 800 μL per 1×10^6 cells. The cells were stored at -80°C until sequencing.
Single-cell mRNA sequencing was performed at Single Cell Discoveries according to standard 10x Genomics 3’ V3.1 chemistry protocol. Prior to loading the cells on the 10x Chromium controller, cells were rehydrated in rehydration buffer. Cells were then counted to assess cell integrity and concentration. Approximately 9,000 cells (comprising 3,000 cells from each of the three layers) from corneas 01, 02, 03 and 04 were separately loaded by layer and cornea. Furthermore, 3,000 cells (1,000 cells from each of the three layers) from corneas 05 and 06, and 3,000 cells from corneas 07 and 08 (from the limbal samples) were separately loaded by cornea. The resulting sequencing libraries were prepared following a standard 10x Genomics protocol.

treatment_protocol: Not applicable to this study.

tag: sample number

value: pooled male and female

tag: age

value: pooled sample (Female - 71 yo + Male - 79 yo)

value: cornea

tag: dissected tissue

value: Limbus pool


title: 04-Epithelium

value: Female

value: Epithelium


title: 04-Stroma

value: Stroma


title: 05-3 layer pool

value: Male

value: 3 layer pool


title: 03-Endothelium

value: Endothelium


title: 03-Stroma


title: 02-Stroma


title: 02-Endothelium


title: 02-Epithelium


title: 01-Stroma