title: SV_PIWI_IP_1

description: small RNA

extract_protocol:
Ribonucleoprotein Immunoprecipitation (RIP)
All steps were performed on ice or at 4 °C unless indicated otherwise. Samples with magnetic beads were placed on magnetic rack for 30sec to ensure beads were pelleted. Dissected tissues were thawed on ice and resuspended in PBS-T (0.05% Tween 20) supplemented with cOmplete, mini EDTA-free protease inhibitor cocktail (Roche Diagnostics, GmbH, Germany) and RNase inhibitor. Samples were transferred to a 60mm cell culture dish (on ice) and placed in a Bio- LinkTMBLX 365 UV-crosslinker. Samples were crosslinked with UV light at 400 mJ/cm2. Samples were then transferred to a fresh 1.5 ml Eppendorf tube. 60 mm dish was then washed once with 500 μl PBS-T and the wash was added to sample. Samples were then centrifuged 8000 x g at 4 °C for 5 min. Supernatant was carefully discarded and samples were resuspended in 250 μl-500 μl RIPA buffer (50mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X 100, 0.1% SDS, 0.1% Na- deoxycholate, 1mM EDTA, 1x cOmplete, mini EDTA-free protease inhibitor cocktail, RNase inhibitor) and transferred to a Wheaton dounce tissue homogenizer chilled on ice. Samples were homogenized 10x with a loose pestle and then 50x with a tight pestle to ensure complete tissue homogenization. The homogenized samples were transferred to a fresh 1.5 ml Eppendorf tube and kept on ice. The homogenizer was rinsed with 250 μl -500 μl IP dilution buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1x cOmplete, mini EDTA-free protease inhibitor cocktail, RNase inhibitor) so as to dilute RIPA buffer 1:1 and the wash solution transferred to same tube as homogenized sample. A 5% aliquot was taken of the lysate to be used as either input for IP-western blot experiments or input for small RNA sequencing experiment. Sample stored at -20 °C.

Meanwhile, Diagenode CHiP-kit protein A magnetic beads were washed before use with 500ul PBS-T and then 500 μl IP buffer (1:1 RIPA buffer and IP dilution buffer). Samples were pre-cleared with pre-washed 25 μl Diagenode CHiP-kit protein A magnetic beads at 4 C with end-to-end rotation for 2 hrs. A second batch of pre-washed Diagenode CHiP-kit protein A magnetic beads were pre-loaded with rabbit-@-PIWI (ab5207) or IP control rabbit IgG: either 4 μg antibody for 25 μl bead slurry for IP-Western blot experiments or with 8 μg antibody for 50 μl bead slurry for IP-targeted proteomics experiment/smallRNA library preparation in 500 μl IP buffer (1:1 RIPA buffer and IP dilution buffer). The bead/antibody solution was incubated at 4  °C for 4-6 hours with end-to-end rotation. Pre-cleared samples were then pelleted on a magnetic rack and supernatant transferred to the preloaded beads (following the disposal of the supernatant). Antibody/preloaded beads/pre-cleared lysate mixture was incubated at 4 °C overnight with end-to-end rotation. The following day, samples were washed 7 x 1 ml wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl2, 10% glycerol, 1% Empigen, RNase inhibitor) 34. Each wash was carefully removed using vacuum suction.  Samples used for IP-western blot or IP-targeted proteomics experiments were eluted with 30 μl Bolt LDS sample buffer (Novex, Life Technologies) supplemented with Bolt sample reducing agent (Novex, Life Technologies) at 95  °C, 1000 rpm for 5min then chilled on ice. Samples were stored at -20 °C until needed. If RNA was needed following PIWI immunoprecipitation, beads were incubated with 100 μl TRI Reagent® for 10 min at room temperature. Input samples were incubated with 10 volumes of TRI Reagent® for 10 min at room temperature.

RNA isolation from beads for small RNA library preparation
Tubes containing phase lock gel were prepared by the filling the lids of 0.2 ml PCR tubes with phase lock gel. The tubes were then quickly spun down. Following incubation with TRI Reagent®, the beads were pelleted using a magnetic rack and the supernatant was transferred to a tube containing phase lock gel. Subsequently, 20 μl chloroform was added, the tubes mixed by shaking and then centrifuged at 3100 x g for 10 min. The upper phase was transferred to a fresh tube containing 50 μl isopropanol and 15 μg GlycoBlueTM, mixed by shaking and then incubated at -20 °C overnight. The samples were then centrifuged at 3100 x g for 10min at 4 °C. The RNA pellet was washed with 100 μl 75% ice cold ethanol, centrifuged at 3100 x g for 10min at 4 °C. Supernatant was carefully decanted off samples and the pellet air dried. The pellet was then resuspended in 4 μl nuclease free water and stored at -80 °C. 5% of lysate used for PIWI IP (25 μl out of 500 μl lysate) was reserved to be used as input for small RNA sequencing experiment. These samples were incubated with 250 μl TRI Reagent® for 10min at room temperature. Subsequently, 50 μl chloroform was added, the tubes mixed by shaking and then centrifuged at 3100 x g for 10min. The upper phase was transferred to a fresh tube containing 125 μl isopropanol and 15 μg GlycoBlueTM, mixed by shaking and then incubated at -20 °C overnight. The samples were then centrifuged at 3100 x g for 10min at 4 °C. The RNA pellet was washed 1x with 250 μl 75% ice cold ethanol, centrifuged at 3100 x g for 10min at 4 °C. Supernatant was carefully decanted off samples and the pellet air dried. The pellet was then resuspended in 6.5 μl nuclease free water and stored at -80 °C.
IP and Input samples processed the same way from now on: The quality and concentration of the RNA samples was analyzed by Agilent Small RNA kit on the Agilent 2100 Bioanalyzer system and NanoDropTM. The RNA samples were then used to make small RNA libraries using the NEBNext Small RNA Library Prep Kit for Illumina (E7330) according to the manufacturer’s instructions. Ligation of the 3`SR adapter was performed at 16 °C for 18 hrs. This longer incubation at a reduced temperature increases ligation efficiency of methylated RNAs such as piRNAs. Since Drosophila RNA is rich in 2S rRNA, a 2S blocking oligo was also used at a final concentration of 0.1 μM to exclude 2S RNA from any downstream reaction. 12 PCR cycles were used to amplify the libraries.
Library preparation was done with NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA) according to the manufacturer instructions with the following minor customizations.  All testicle samples, but not sperm samples, were downscaled to half volume, using 3 μl of input RNA instead of 6 ul as recommended. In all steps the primers in the kit were diluted 1:3 prior to use. 2S rRNA was blocked by adding anti-sense oligos (5´-TAC AAC CCT CAA CCA TAT GTA GTC CAA GCA372 SpcC3 3´; 10 µM) and set to hybridized in the same step as NEBNext SR RT primer (pink). Amplification was made during 16 cycles and amplified libraries were cleaned using Agencourt AMPure XP (Beckman Coulter, Brea, CA) and size selected for 130 to 165 nt fragments on a pre-casted 6% polyacrylamide Novex TBE gel (Invitrogen, Waltham, MA). Gel extraction was done using Gel breaker tubes (IST Engineering, Milpitas, CA) in the buffer provided in the NEBNext kit. Disintegrated gels were incubated at 37 °C for 1 hour on a shaker, quickly frozen for 15 minutes at −80 °C, followed by another incubation for 1 hour. Any remaining gel debris was removed by Spin-X 0.45 μm centrifuge tubes (Corning Inc., Corning, NY) as recommended by the NEBnext protocol. The libraries were precipitated overnight at −80 °C by adding 1 μl of GlycoBlue (Invitrogen) as co-precipitant, 0.1 times the volume of Acetate 3M (pH 5.5), and 3 times the volume of 100% ethanol. Library concentrations were estimated using QuantiFluor ONE ds DNAsystem on a Quantus fluorometer (Promega, Madison, WI). Pooled libraries were sequenced on NextSeq 500 with NextSeq 500/550 High Output Kit version 2, 75 cycles (Illumina, San Diego, CA). All pooled libraries passed Illumina’s default quality control.

value: small RNA


title: Mo.6C+.IL4.Sp#2

status: Public on Jan 01 2022

type: SRA

city: Boston

first: Liang

last: Yang

state: MA

address: 77 Avenue Louis Pasteur

department: Pathology

country: USA

institute: Harvard Medical School

library_source: transcriptomic

data_processing:
Reads were mapped to the mouse reference genome (mm10) with STAR 2.7.3a
Non-uniquely mapped reads were discarded.
The uniquely mapped reads were assigned to genes using featureCounts with GENCODE annotation (vM25)
Genome_build: mm10
Supplementary_files_format_and_content: raw read counts

molecule: total RNA

organism: Mus musculus

source_name: Monocyte

extract_protocol:
1,000 Cells were double-sorted by FACS Aria 561 into 5ul Buffer TCL (Qiagen) with 1% 2-Mercaptoethanol (Sigma).Total RNA was captured and purified on RNAClean XP beads (Beckman Coulter)
total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3) and converted to cDNA via reverse transcription. First-strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were then PCR amplified for 18 cycles using barcoded primers such that each sample carried a specific combination of eight-base Illumina P5 and P7 barcodes and were pooled together prior to Smart sequencing. Smart-seq paired-end sequencing was performed on an Illumina NextSeq500 using 2 x 38bp reads with no further trimming.

treatment_protocol: 5-8 week old C57Bl/6J mice shipped from the Jackson Laboratory, Bar Harbor. Mice were injected retro-orbitally with either a free cytokine or a cytokine-antibody complex with a 2 hour acute readout. Further, mice were injected with 10ug mIL2/110uL PBS,5 μg IL-4 with 25 μg 11B11 in 110 uL PBS, 0.75ug mIL-7 to 15ug anti-m/h IL-7 M25 mAb  in 110uL PBS, 5ug mIL9 in 110uL PBS, 2.5ug mIL-15 to 15ug mIL15Ra-Fc in 110uL PBS, or 20ug mIL-21 in 110uL PBS.

tag: strain

tag: tissue

value: Spleen

tag: cell type

value: Monocyte


title: RNA_KO2_3

city: Montreal

first: Michael

last: Witcher

state: Quebec

address: 3755 Chem. de la Côte-Sainte-Catherine

department: Experimental Medicine

country: Canada

institute: McGill University

data_processing:
Reads were trimmed with Trimmomatics (Bolger et al. 2014) using the following condition: ILLUMINACLIP:$Adapters:2:30:10:8:true, HEADCROP:4, SLIDINGWINDOW:4:30, LEADING:3, TRAILING:3, MINLEN:30.
Alignment on hg19 human genome was performed with STAR 2.5.4b (Dobin et al. 2013) default parameters, and converted into bam format using Samtools 1.9 (Li et al. 2009).
Differential expression analysis was generated using FeatureCounts count matrix (Liao et al. 2014)
Genome_build: hg19
Supplementary_files_format_and_content: Count Matrix from featureCount

organism: Homo sapiens

source_name: CTCF +/-  #2 MCF10A

growth_protocol: MCF10A cell line and mutants were maintained in DMEM/F12 50/50 (Wisent, #319-085-CL) supplemented with EGF (100μg/ml, Wisent, #511-110-UM), Insulin (10mg/ml, Wisent, #H511-016-U6), Hydrocortisone (1mg/ml, Sigma, #H0888-1G), Horse Serum (2%, Wisent, #065150) and Choleratoxin (1mg/ml, Sigma, #C8052-2MG) in an incubator at 37C and 5% CO2.

extract_protocol:
Total RNA was extracted according to Sigma RNA Extraction Kit (Sigma, #RTN350-1KT) protocol. RNA quantity and quality was measured using Nanodrop.
RNA was sent to Genome Quebec for polyA RNA library preparation using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina

value: Breast

tag: genotype

value: CTCF +/-


title: Mo.6C+.IL4.Sp#1


title: RNA_KO2_2


title: WT2U_ZT44

city: New York

first: Zikun

last: Wang

state: New York

address: 1230 York Av

institute: The Rockefeller University

data_processing:
bcl2fastq v2.20 was used for basecalling.
STAR v2.4.2a was used for alignment.
featureCounts was used for generate counts matrix.
Genome_build: FlyBase release 6.13 (May 2016)
Supplementary_files_format_and_content: tab-delimited text files include raw counts for each aligned gene for each sample

organism: Drosophila melanogaster

source_name: fly heads

extract_protocol:
Total RNA was extracted using TRIzol reagents and homogenized using a BeadBug microtube homogenizer (Benchmark Scientific). Samples were further extracted using chloroform and the aqueous phase containing nucleic acid was collected. RNeasy Mini Kit (Qiagen) was then used to remove DNA with DNase and further purify the samples, following the manufacturer's protocol.
Sequencing libraries were prepared with the Illumina TruSeq stranded mRNA LT kit

value: Canton-S w1118 (iso1CJ)

tag: age

value: 27 days old

tag: timepoint

value: Zeitgeber time 44


title: 53SE_s1

city: Beijing

first: shaofang

last: Li

state: Beijing

address: No 50, ZhanghuaLu, HAIDIAN

department: Beijing Vegetable Research Center

country: China

institute: Beijing Academy of Agriculture and Forestry Science

library_source: genomic

data_processing:
library strategy: Whole genome sequencing
All sequenced reads were and mapped back toKittaake V3 genome using BWA mem
SNVs  and indels were called on each genome data using GATK, Strelka, LoFreq, separately
Genome_build: Kittaake V3; https://phytozome-next.jgi.doe.gov/info/OsativaKitaake_v3_1
Supplementary_files_format_and_content: GATK.vcf.gz are mutations files called by GATK,Strelka.vcf.gz are mutations called by Strelka, LoFreq.vcf are mutations called by LoFreq

molecule: genomic DNA

organism: Oryza sativa

source_name: 53SE_four-week plants

growth_protocol: Plants were grown with 16hr of light at  28 °C daytime,  25°C  night

extract_protocol:
Genomic DNA was extracted using CTAB methods and treated with RNase I
All libraryies were built using NEB Next® UltraTM DNA Library Prep Kit

tag: cultivar

value: Kitaake

tag: age/tissue

value: four-week plants


title: 53SE_s2


title: INC1_ZT20

value: Zeitgeber time 20


title: Mo.6C+.IL21.Sp#2


title: RNA_KO2_1


title: Mo.6C+.IL21.Sp#3


title: Mo.6C+.IL21.Sp#1


title: WT2U_old_ZT20

value: 49 days old


title: RNA_KO1_3

source_name: CTCF +/-  #1 MCF10A


title: Mo.6C+.IL2.Sp#3


title: RNA_KO1_2


title: WT2U_old_ZT32

value: Zeitgeber time 32


title: WAKED2_ZT20


title: WT2U_ZT20


title: RNA_KO1_1


title: 50bg_s2

source_name: 50bg_four-week plants


title: 50bg_s3


title: INC1_ZT32


title: 50DEP_s3

source_name: 50DEP_four-week plants


title: 50DEP_s2


title: WAKED2_ZT32


title: Mo.6C+.IL2.Sp#1


title: RNA_WT_3

source_name: WT MCF10A

value: WT


title: WT2U_ZT32


title: Mo.6C+.IL15.Sp#3


title: RNA_WT_2


title: 50G_s3

source_name: 50G_four-week plants


title: WT2U_old_ZT8

value: Zeitgeber time 8


title: Mo.6C+.IL15.Sp#2


title: Mo.6C+.IL2.Sp#2


title: WAKED2_ZT8


title: 50bg_s1


title: 50G_s2


title: RNA_WT_1


title: INC1_ZT8


title: MF.RP.PBS.Sp#6

source_name: Red pulp Macrophage

value: Red pulp Macrophage


title: H3K27ac_KO_Input

data_processing:
Reads were trimmed with Trimmomatics (Bolger et al. 2014) using the following parameters: ILLUMINACLIP:$Adapters:2:30:10, LEADING:30, TRAILING:30, SLIDINGWINDOW:4:30, MINLEN:30.
Alignment on hg19 human genome was performed using BWA (Li et al. 2009) default conditions.
Sam files generated by BWA were converted to bam format using Samtools (Li et al. 2009).
Peak calling was performed with MACS2 (Zhang et al. 2008) default condition and normalization on the respective Input dataset of each cell lines.
Bigwig files used for visualization were generated from the fragment pileup bedGraph using the BedGraphToBigwig function.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files

source_name: CTCF +/- MCF10A

extract_protocol:
70-80% confluent cells were fixed 10 minutes in 4% formaldehyde and stored at -80C. The pellets were subsequently resuspended in 1ml of ChIP-buffer [0.25% NP-40, 0.25% Triton X-100, 0.25% Sodium Deoxycholate, 0.005% SDS, 50nM Tris (pH8), 100mM NaCl, 5mM EDTA, 1X PMSF, 2mM NaF, 1X P8340 Cocktail Inhibitor (Roche)] and sonicated with a probe sonicator (Fisher Scientific Sonic Dismembrator Model 500) using the following cycles: 5 cycles at 20% power, 5 cycles at 25% power, and 5 cycles at 30% power. Each cycle lasts 10 seconds, and the samples are kept on ice between each cycle to avoid overheating. Next, the samples are spun at high speed in a microcentrifuge for 30 minutes. Then, lysates are collected and protein concentration measured using the Bradford assay, as described above. Based on protein concentrations, samples are diluted to 2mg/ml proteins in ChIP-buffer and 50ul/ml of Protein G Plus-Agarose Suspension Beads (Calbiochem, IP04-1.5ML) are added for 3h to preclear. 2% of the sample is collected as input and kept at -20 °C until DNA purification. Immunoprecipitation is carried out at 4°C overnight with 1ml of sample, 60ul of beads and primary antibody. The beads are then washed once with Wash1, Wash2, Wash3 [0.10% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris (pH 8), 150/200/500mM NaCl for Wash 1,2,3 respectively], Wash LiCl [0.25M LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1mM EDTA, 10mM Tris (pH8)] and twice with TE buffer [10mM Tris (pH8), 1mM EDTA]. Then, beads are resuspended in elution buffer [1% SDS, 0.1M NaHCO3]. The samples are decrosslinked overnight at 65 °C. 20µg of Proteinase K (Sigma, # 39450-01-6) is added for 1h at 42 °C. Then, DNA is purified using BioBasic DNA collection column (BioBasic, #SD5005). DNA concentration was assessed via Picogreen assay (Invitrogen, #P7589).
Library were preapred at Genome Quebec (for CTCF and H3K4me3) or The Centre for Applied Genomics (TCAG) at SickKids Hospital (for H3K27ac) using  NEB Ultra II DNA kit (no shearing).


title: H3K27ac_WT_Input


title: Mo.6C+.IL15.Sp#1


title: MF.RP.PBS.Sp#5


title: WT2U_old_ZT40

value: Zeitgeber time 40


title: 50G_s1


title: 50SE_s3

source_name: 50SE_four-week plants


title: WT2U_ZT8


title: H3K4me3_KO_Input


title: H3K4me3_WT_Input


title: 50SE_s1


title: MF.RP.PBS.Sp#3


title: 50DEP_s1


title: CTCF_KO_Input


title: WAKED2_ZT40


title: Cas_s3

source_name: Cas_four-week plants

value: Cas


title: WT2U_old_ZT16

value: Zeitgeber time 16


title: MF.RP.PBS.Sp#1

value: Spleen


title: Cas_s2


title: CTCF_WT_Input

value: Breast


title: H3K27ac_KO_2

value: CTCF +/-


title: MF.RP.PBS.Sp#4


title: INC1_ZT16


title: 49bg_s3

source_name: 49bg_four-week plants


title: H3K27ac_WT_2


title: MF.RP.IL7.Sp#3


title: MF.RP.IL7.Sp#1


title: Cas_s1


title: 50SE_s2


title: 49bg_s2


title: H3K27ac_KO_1


title: MF.RP.IL4.Sp#3


title: WT2U_old_ZT28

value: Canton-S w1118 (iso1CJ)

value: Zeitgeber time 28


title: MF.RP.IL4.Sp#2


title: INC1_ZT40

value: 27 days old


title: H3K4me3_KO_2


title: 49bg_s1

value: Kitaake


title: H3K27ac_WT_1


title: INC1_ZT28


title: MF.RP.PBS.Sp#2


title: MF.RP.IL21.Sp#3


title: H3K4me3_KO_1


title: WAKED2_ZT28


title: 49bTm_s2

source_name: 49bTm_four-week plants

value: four-week plants


title: MF.RP.IL21.Sp#2


title: WAKED2_ZT16


title: 49bTm_s1


title: INC1_ZT4

value: Zeitgeber time 4


title: MF.RP.IL15.Sp#2


title: WT2U_old_ZT4

value: 49 days old


title: WT2U_ZT16


title: H3K4me3_WT_1


title: 49bM_s2

source_name: 49bM_four-week plants


title: MF.RP.IL15.Sp#3


title: CTCF_KO_1


title: WAKED2_ZT4


title: WT2U_ZT4


title: 49bM_s3


title: 49bM_s1


title: MF.RP.IL15.Sp#1


title: CTCF_KO_2


title: H3K4me3_WT_2


title: 49bTm_s3


title: 49bAG_s4

source_name: 49bAG_four-week plants


title: WT2U_old_ZT36

value: Zeitgeber time 36


title: MF.PBS.PC#12

source_name: Macrophage

value: Peritoneal Cavity

value: Macrophage


title: CTCF_WT_4

value: WT


title: MF.PBS.PC#13


title: CTCF_WT_3


title: 49bAG_s3


title: CTCF_WT_2


title: WAKED2_ZT36


title: MF.PBS.PC#10


title: 49bAG_s2


title: INC1_ZT36


title: WT2U_ZT36


title: MF.PBS.PC#9


title: 49bAG_s1


title: MF.PBS.PC#8


title: WT2U_old_ZT12

value: Zeitgeber time 12


title: HiC_KO2_2

data_processing:
Raw sequencing read were mapped, filtered, and binned using the runHiC pipeline (Wang et al. 2016).
Contact matrix were binned at 5kb and 10kb resolution and stored in “.cool” format. (5kb matrices will be made available on demand)
Genome_build: hg19
Supplementary_files_format_and_content: 10k Interaction matrices stored in cool format

extract_protocol:
Hi-C data was generated from 1M WT, CTCF +/- #1 or CTCF +/- #2 MCF10A cells per replicates, with 2 replicates per conditions, using the Arima-HiC kit, according to the manufacturer’s protocols (Arima Genomics).
Library preparation was performed using KAPA Hyper Prep Kit  (#07962312001) following Arima protocol for library preparation.


title: INC1_ZT12


title: WAKED2_ZT12


title: 46bg_s2

source_name: 46bg_four-week plants


title: MF.PBS.PC#7


title: HiC_KO1_1


title: HiC_KO1_2


title: WT2U_ZT12

source_name: fly heads


title: 46bg_s1


title: MF.PBS.PC#6


title: 46bM_s3

source_name: 46bM_four-week plants


title: MF.PBS.PC#5


title: HiC_WT_2


title: MF.PBS.PC#11


title: WT2U_old_ZT24

value: Zeitgeber time 24


title: MF.PBS.PC#4


title: 46bM_s2


title: CTCF_WT_1


title: INC1_ZT24


title: 46bM_s1


title: HiC_KO2_1

source_name: CTCF +/-  #1 MCF10A


title: 46bg_s3


title: 46bGG_s3

source_name: 46bGG_four-week plants


title: 46bGG_s2


title: HiC_WT_1


title: MF.IL4.PC#3


title: B6_B_R1

city: Stanford

first: Giovanni

middle: Charles

last: Forcina

state: California

address: 327 Campus Drive

department: Biology

institute: Stanford University

description:
cancer derived cell line
B6_B_R1

data_processing:
Basecalls performed using CASAVA
Reads were filtered using the following criteria (from NovoGene manual): (1) Remove reads containing adaptors;  (2) Remove reads containing N > 10% (N represents base that could not be determined);  (3) The Qscore (Quality value) of over 50% bases of the read is <= 5.  RNA-seq Adapter information:  NEBNext® Ultra  TM RNA Library Prep Kit  RNA 5' Adapter(5'Adaptor)  5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT  RNA 3' Adapter(3'Adaptor,The underlined 6bp bases is Index)  5' GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG
Paired-end reads were mapped to the genome using STAR v2.5
Quantification of gene expression level (from NovoGene manual): "HTSeq v0.6.1 was used to count the read numbers mapped of each gene. And then FPKM of each   gene was calculated based on the length of the gene and reads count mapped to this gene.'"
Differential expression of genes was calculated using DESeq2 R package (2_1.6.3)
Genome_build: homo sapiens hg19
Supplementary_files_format_and_content: The processed .txt files contain the readcounts and FPKM of each gene for the conditions.

source_name: A549 cells

growth_protocol: A549 cells were grown in DMEM (+ pen/strep and 10% FBS). Cells were split every 2-3 days and typically seeded at 1,000,000 cells in a T75 flask.

extract_protocol:
Cells were scraped into 0.5 mL of PBS, spun down, and then RNA was extracted using the Qiagen Rneasy (cat. 74134) kit as per manufacturer's instructions.
As per Novogene manual: "Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is  subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded  cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to  prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter,  Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads."

treatment_protocol: Cells were treated with DMSO vehicle control or bortezomib (200 nM) for 4 h.

tag: cell line

tag: treatment

value: bortezomib treated

value: NFE2L1 KO2


title: WT2U_ZT24


title: MF.PBS.PC#3


title: B6_B_R2

description:
cancer derived cell line
B6_B_R2


title: WT2U_old_ZT0

value: Zeitgeber time 0


title: INC1_ZT0


title: MF.IL4.PC#2


title: 46bAG_s3

source_name: 46bAG_four-week plants


title: 46bAG_s2


title: B6_D_R1

description:
cancer derived cell line
B6_D_R1

value: vehicle treated


title: 46bGG_s1


title: B4_B_R1

description:
cancer derived cell line
B4_B_R1

value: NFE2L1 KO1


title: MF.IL4.PC#1


title: WT2U_ZT0

value: Canton-S w1118 (iso1CJ)


title: 46bAG_s1


title: WAKED2_ZT0

value: 27 days old


title: MF.IL21.PC#2


title: MF.PBS.PC#2


title: B4_D_R2

description:
cancer derived cell line
B4_D_R2


title: WAKED2_ZT24


title: MF.IL21.PC#1


title: MF.PBS.PC#1


title: OL95-2

city: Marburg

first: Muriel

last: van Teeseling

address: Hans-Meerwein-Strasse 4

country: Germany

institute: Philipps-Universität Marburg

data_processing:
Base-calling was perform online during the sequencing procedure with the Real-Time Analysis (RTA) software version 2.4.11 and System Suite Version 2.0.2.2
Quality and adapter trimming was performed with Timmomatic v0.36:   SE  -threads 24  -phred33  ILLUMINACLIP:TruSeq3-vertis.fa:2:30:10 (contains Illumina TruSeq adapter sequences and a 80 nt langen polyA-tail)  SLIDINGWINDOW:4:15  LEADING:3  TRAILING:3  MINLEN:30
Mappings against reference CP000158 were performed with bowtie2 with the --local parameter
Expression values were calculated using FeatureCounts with the following parameters:  Feature type  -t gene  Attribute type  -g gene_id  Strand type  -s 1 (fwd)  Feature level  -f (yes)  Multi-mapping reads will be counted  -M (yes)  fractional count 1/n  --fraction (yes)  Minimum overlap  --minOverlap 30
Genome_build: CP000158
Supplementary_files_format_and_content: RPKMs

organism: Hyphomonas neptunium

growth_protocol: The indicated strains were grown to the mid-exponential growth phase.

extract_protocol:
Total RNA was isolated from the cell pellets using a bead mill and the mirVana RNA isolation kit (Ambion) including DNase treatment. From the total RNA samples, ribosomal RNA molecules were depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre).
From the rRNA-depleted RNA samples, first-strand cDNA was synthesized using a N6 randomized primer. After fragmentation, the Illumina TruSeq sequencing adapters (containing barcode sequences) were ligated in a strand-specific manner to the 5' and 3' ends of the cDNA fragments. The cDNA was finally amplified with PCR using a proof-reading enzyme. For Illumina NextSeq sequencing, the cDNAs were pooled in approximately equimolar amounts. The cDNA pool was eluted from a preparative agarose gel in the size range of 200 – 500 bp. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina, yielding a total length of the flanking sequences of 122 bp.

treatment_protocol: After dilution of each culture to an OD580 of 0.15-0.2, the cells were washed two times in MB medium, harvested by centrifugation, snap-frozen in liquid nitrogen and stored at -80°C.

value: {delta}divK mutant


title: Mock DNA

city: Seattle

first: Dmitry

last: Sutormin

state: WA

address: 401 Terry Ave N

institute: Institute for Systems Biology

description: Exponentially growing culture (OD600=0.6) in LB was crosslinked with 1% formaldehyde. Formaldehyde was quenched by incubation with 0.25 M glycine. Cells were harvested by centrifugation. Cell pellets were washed twice with TBS, resuspended in ChIP Lysis Buffer 1, followed by addition of 2x ChIP Lysis Buffer 2, containing RNaseA. Resuspended cells were pre-lysed by the addition of 1 µg of Lysozyme and incubated at 37°C for 10 min, followed by sonication in a 2-ml Eppendorf tube in an ice-water bath to achieve a maximum yield of 300-400 bp fragments. Lysates were clarified by centrifugation at 13000 g for 10 min at 4°C, and the resulting supernatant was used for further analysis. 100 µL aliquot of the clarified lysate was first treated with proteinase K (Thermo-Fisher Scientific) at 0.125 mg/ml in the presence of 1% SDS for 4 h at 37°C followed by DNA de-crosslinking by incubation at 65°C for 6 h. The Mock-DNA material was purified using Qiagen PCR purification kit (Qiagen) and quantified by Nano-Drop spectrophotometer. The DNA fragment size distribution was assessed by DNA electrophoresis. A 50-100 ng of Mock-DNA were end repaired by a mix of T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow DNA polymerase (NEB) and purified by Qiaquick PCR DNA purification kit (Qiagen). The eluted DNA material was A-tailed by Klenow Fragment (3ʼ to 5ʼ exo minus) (NEB) followed by purified by MinElute PCR purification Kit (Qiagen). Illumina Multiplex Adapters (MPA) were ligated with Quick DNA ligase (NEB) and DNA was purified by AMPure XP beads (Beckman Coulter). Resulting library was separated by agarose electrophoresis with following size selection of DNA bands corresponding to 220 bp, which were excised by Gel X-tracta tool (USA scientific) and purified with Gel Extraction Kit (Qiagen). The purified DNA material was PCR-amplified 18 cycles using Phusion polymerase (NEB) and purified by MinElute PCR purification kit (Qiagen). DNA sequencing was performed by Illumina HiSeq 50+50 bp paired-end protocol.

data_processing:
Raw reads were trimmed and filtered using Trimmomatic (ILLUMINACLIP:2:30:10 LEADING:0 TRAILING:0 SLIDINGWINDOW:4:0 MINLEN:30).
Processed reads were aligned to the E. coli w3110 MuSGS genome with BWA MEM (default settings).
Resulting sam files were converted to bam and sorted using samtools.
Sequencing coverage depth was calculated from bam file using samtools and converted to wig format using custom script (see https://github.com/sutormin94/TopoI_ChIP-Seq).
Coverage depth fold enrichment of IP sample over Mock sample was calculated using custom script.
Peaks were identified by setting the fold enrichment threshold.
Genome_build: NC_007779.1_w3110_Mu (custom build, fasta and genome annotation are provided as supplementary files)
Supplementary_files_format_and_content: wig files with sequencing coverage depth.
Supplementary_files_format_and_content: wig files with sequencing coverage depth fold enrichment of IP sample over Mock sample.
Supplementary_files_format_and_content: tab-delimeted files with enrichment peaks coordinates.

organism: Escherichia coli

source_name: Escherichia coli DY330 rpoC-TAP

growth_protocol: For TAP-tagged RNAP-ChIP, we used E. coli DY330 strain (Dharmacon) carrying chromosomally TAP-tagged rpoC gene. Cell culture (V=200ml) grown to mid-exponential phase OD600~0.5-0.7 in LB containing 50 µg/ml of kanamycin.

extract_protocol:
For the Mock-DNA fragment size analysis, a 100 µl aliquot of the clarified lysate was first treated with proteinase K (Thermo-Fisher Scientific) at 0.125 mg/ml in the presence of 1% SDS for 4 h at 37°C followed by DNA de-crosslinking by incubation at 65°C for 6 h. The Mock-DNA material was purified using Qiagen PCR purification kit (Qiagen) and quantified by Nano-Drop spectrophotometer. The DNA fragment size distribution was assessed by DNA electrophoresis. For the preparation of RNAP-ChIP DNA, the remaining lysate (~900 µl) was purified by immunoprecipitation (IP). The lysate was mixed with 10 µl of IgG-agarose (GE Healthcare) and incubated at 4°C overnight on a rotating mixer in the presence of Protease Inhibitor Cocktail (Sigma). Then affinity resin was washed consecutively with 1 ml each of the following solutions: 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 2 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 1 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 200 mM NaCl; and twice with RIPA buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 1% NP-40, 0.1% Deoxycholate, 1% SDS). All wash procedures were carried out by resuspending the beads in the wash solution and inverting the tube several times, followed by brief centrifugation and removing the supernatant by vacuum aspiration. The last wash was done for 20 min at 4°C on a rotary mixer. The RNAP-DNA crosslinks were eluted by incubation with ChIP Elution Buffer (10 mM Tris pH 8.0, 30 mM EDTA, 0.1% SDS) at 65°C for 4 hours (or O/N) on a shaker at 800-900 rpm. The resulting material was treated with Proteinase K (Thermo Fisher Scientific) (0.2 mg/ml) at 37°C for 3-5 h, followed by IP-DNA decrosslinking by incubation at 95°C for 2h. The IP-DNA was finally purified using ChIP DNA Cleaning & Concentrator kit (Zymo Research).
A 50-100 ng of IP-DNA or Mock-DNA were end repaired by a mix of T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow DNA polymerase (NEB) and purified by Qiaquick PCR DNA purification kit (Qiagen). The eluted DNA material was A-tailed by Klenow Fragment (3ʼ to 5ʼ exo minus) (NEB) followed by purified by MinElute PCR purification Kit (Qiagen). Illumina Multiplex Adapters (MPA) were ligated with Quick DNA ligase (NEB) and DNA was purified by AMPure XP beads (Beckman Coulter). Resulting library was separated by agarose electrophoresis with following size selection of DNA bands corresponding to 220 bp, which were excised by Gel X-tracta tool (USA scientific) and purified with Gel Extraction Kit (Qiagen). The purified DNA material was PCR-amplified 18 cycles using Phusion polymerase (NEB) and purified by MinElute PCR purification kit (Qiagen). DNA sequencing was performed by Illumina HiSeq 50+50 bp paired-end protocol.

treatment_protocol: The culture was crosslinked by adding formaldehyde to a final concentration of 1%, followed by incubation for 20 min at room temperature, with agitation. Crosslinking was stopped by the addition of sterile glycine to a final concentration of 0.25 M. Cells were incubated for 20 min at room temperature and harvested by centrifugation at 3000 g, 10 min, 4°C. Cell pellets were washed twice with 20 ml of ice-cold 20 mM Tris-HCl pH 7.6, containing 60 mM NaCl (TBS), resuspended in 0.5 ml of ChIP Lysis Buffer 1 (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM EDTA, 20% Sucrose), followed by addition of 0.5 ml of 2x ChIP Lysis Buffer 2 (200 mM Tris-HCl pH 8.0, 600 mM NaCl, 4% Triton X-100), containing RNaseA (1 µg/ml). Resuspended cells were pre-lysed by the addition of 1 µg of Lysozyme and incubated at 37°C for 10 min, followed by sonication in a 2-ml Eppendorf tube in an ice-water bath to achieve a maximum yield of 300-400 bp fragments. Lysates were clarified by centrifugation at 13000 g for 10 min at 4°C, and the resulting supernatant was used for further analysis.

value: DY330 rpoC-TAP

tag: growth phase

value: Exponential phase

tag: optical density at 600 nm

tag: ip or mock

value: Mock DNA


title: B4_D_R1

description:
cancer derived cell line
B4_D_R1


title: B4_B_R2

description:
cancer derived cell line
B4_B_R2


title: OL95-1


title: MF.IL2.PC#3


title: Mock control, non-induced control, replicate 3

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 3.

data_processing:
Library strategy: Topo-Seq
Raw reads were trimmed and filtered using Trimmomatic (ILLUMINACLIP:2:30:10 LEADING:0 TRAILING:0 SLIDINGWINDOW:4:0 MINLEN:30).
Processed reads were aligned to the E. coli w3110 MuSGS genome with BWA MEM (default settings).
Strand-specific coverage for forward and reverse strands were obtained based on read alignments stored in SAM files using custom script (see https://github.com/sutormin94/TopoI_Topo-Seq).
Genome_build: NC_007779.1_w3110_Mu (custom build, fasta and genome annotation are provided as supplementary files)
Supplementary_files_format_and_content: wig files with strand-specific sequencing coverage depth.

source_name: Escherichia coli DY330 topA-SPA pBAD33 topA_strepII G116S M320V

growth_protocol: For starter culture, a single colony of E. coli DY330 topA-SPA harboring the pBAD33 topA_strepII G116S M320V plasmid was inoculated in 5 mL of LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose. The starter was grown overnight at 37C with shaking 180 rpm. 1 mL of the starter was inoculated in 100 mL of fresh LB supplemented with the antibiotic and glucose, and the culture was cultivated at 37C with shaking 180 rpm until reaching OD600=0.4.

extract_protocol:
For Input DNA (induced and non-induced mock samples) 100 ul of the lysates were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). DNA fragmentation range was assessed by electrophoresis. For cleavage complexes immunoprecipitation (induced and non-induced IP samples), 900 ul of the remaining lysates were mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter).
NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. DNA sequencing was performed using Illumina NextSeq with 150+150 bp paired-end protocol. Libraries preparation and sequencing were performed at Skoltech Sequencing Core Facilities. Topo-Seq experiments were performed in triplicate.

treatment_protocol: When OD600 of the culture reached 0.4, the culture (100 mL) was bisected and arabinose was added (final concentration 10 mM) to one half (50 mL) to induce expression of EcTopoI G116S M320V. Then, cultivation proceeded for 30 min (final OD600~0.6) and cells from induced and non-induced samples were harvested by centrifugation (3000 g, 5min, 4C). Cells pellets were snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysates were clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis.

value: DY330 topA-SPA

tag: plasmid

value: pBAD33 topA_strepII G116S M320V

tag: induction

tag: ip or control

value: Mock control


title: B2_B_R2

description:
cancer derived cell line
B2_B_R2


title: MF.IL2.PC#2

value: Peritoneal Cavity

value: Macrophage


title: OL94-2

value: {delta}divJ mutant


title: B2_B

description:
cancer derived cell line
B2_B

value: bortezomib treated


title: MF.IL2.PC#1


title: OL94-1


title: Mock control, non-induced control, replicate 2

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 2.


title: OL20-2

value: {delta}pleC mutant


title: Mock control, non-induced control, replicate 1

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 1.


title: B2_D_R2

description:
cancer derived cell line
B2_D_R2


title: B2_D

description:
cancer derived cell line
B2_D


title: OL20-1


title: MF.IL15.PC#3


title: MF.IL15.PC#2


title: Mock control, EcTopoI G116S M320V induced, replicate 3

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 3.

value: TopoI G116S M320V expression induced with 10 mM arabinose


title: Mock control, EcTopoI G116S M320V induced, replicate 2

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 2.


title: MF.IL15.PC#1


title: WT-2

source_name: WT

value: wild-type


title: HCT116 - C-1 rep 2

first: Tomasz

last: Sewastianik

address: 450 Brookline Ave

department: Oncologic Pathology

institute: Dana-Farber Cancer Institute

data_processing:
Sequencing reads were processed as described in Cornwell, M. I. et al. BMC Bioinformatics 19, 1–14 (2018)
Genome_build: hg38
Supplementary_files_format_and_content: CSV text file with TPM values for every sample

growth_protocol: Cells were cultured in McCoy’s 5A supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ºC with 5% CO2 in a humidified incubator.

extract_protocol:
RNA was extracted using TRIZOL.
Libraries were prepared using Roche Kapa stranded mRNA HyperPrep sample preparation kits from 200ng of purified total RNA according to the manufacturer’s protocol.

treatment_protocol: Cells were treated in triplicate with the DMSO vehicle control or 20µM of inhibitor for 48h.

tag: tumor type

value: colorectal cancer


title: WT-1

tag: strain


title: HCT116 - C-1 rep 3


title: OL161-ind-2

growth_protocol: The indicated strains were grown for 24 h in the absence of presence (0.3 mM) CuSO4

value: conditional cckAmutant

tag: supplement

value: 0.3 mM CuSO4


title: MF.IL9.PC#1

tag: tissue

tag: cell type


title: MF.IL9.PC#2


title: RpoC ChIP-Seq

description: Exponentially growing culture (OD600=0.6) in LB was crosslinked with 1% formaldehyde. Formaldehyde was quenched by incubation with 0.25 M glycine. Cells were harvested by centrifugation. Cell pellets were washed twice with TBS, resuspended in ChIP Lysis Buffer 1, followed by addition of 2x ChIP Lysis Buffer 2, containing RNaseA. Resuspended cells were pre-lysed by the addition of 1 µg of Lysozyme and incubated at 37°C for 10 min, followed by sonication in a 2-ml Eppendorf tube in an ice-water bath to achieve a maximum yield of 300-400 bp fragments. Lysates were clarified by centrifugation at 13000 g for 10 min at 4°C, and the resulting supernatant was used for further analysis. 900 µL of the lysate were mixed with 10 µl of IgG-agarose (GE Healthcare) and incubated at 4°C overnight on a rotating mixer in the presence of Protease Inhibitor Cocktail (Sigma). Then affinity resin was washed consecutively with 1 ml each of the following solutions: 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 2 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 1 M NaCl; 40 mM Tris-HCl pH 7.9, 0.5% Tween-20, 200 mM NaCl; and twice with RIPA buffer (50 mM Tris-HCl pH 7.4, 140 mM NaCl, 1% NP-40, 0.1% Deoxycholate, 0.1% SDS). All wash procedures were carried out by resuspending the beads in the wash solution and inverting the tube several times, followed by brief centrifugation and removing the supernatant by vacuum aspiration. The last wash was done for 20 min at 4°C on a rotary mixer. The RNAP-DNA crosslinks were eluted by incubation with ChIP Elution Buffer (10 mM Tris pH 8.0, 30 mM EDTA, 1% SDS) at 65°C for 4 hours (or O/N) on a shaker at 800-900 rpm. The resulting material was treated with Proteinase K (Thermo Fisher Scientific) (0.2 mg/ml) at 37°C for 3-5 h, followed by IP-DNA decrosslinking by incubation at 95°C for 2h. The IP-DNA was finally purified using ChIP DNA Cleaning & Concentrator kit (Zymo Research). A 50-100 ng of IP-DNA were end repaired by a mix of T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow DNA polymerase (NEB) and purified by Qiaquick PCR DNA purification kit (Qiagen). The eluted DNA material was A-tailed by Klenow Fragment (3ʼ to 5ʼ exo minus) (NEB) followed by purified by MinElute PCR purification Kit (Qiagen). Illumina Multiplex Adapters (MPA) were ligated with Quick DNA ligase (NEB) and DNA was purified by AMPure XP beads (Beckman Coulter). Resulting library was separated by agarose electrophoresis with following size selection of DNA bands corresponding to 220 bp, which were excised by Gel X-tracta tool (USA scientific) and purified with Gel Extraction Kit (Qiagen). The purified DNA material was PCR-amplified 18 cycles using Phusion polymerase (NEB) and purified by MinElute PCR purification kit (Qiagen). DNA sequencing was performed by Illumina HiSeq 50+50 bp paired-end protocol.

value: IP


title: OL161-ind-1

tag: genotype


title: MF.IL7.PC#3


title: HCT116 - DMSO rep 2


title: Topo-Seq, non-induced control, replicate 3

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 3.


title: Topo-Seq, non-induced control, replicate 2

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 2.


title: MF.IL7.PC#2


title: Topo-Seq, non-induced control, replicate 1

description: Exponentially growing culture (OD600=0.6, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 1.


title: HCT116 - DMSO rep 3


title: HCT116 - DMSO rep 1


title: OL161-unind-2


title: OL161-unind-1


title: MF.IL7.PC#1


title: Mock control, EcTopoI G116S M320V induced, replicate 1

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For Input DNA, 100 ul of the lysate were treated with proteinase K (Thermo-Fisher Scientific) for 2 h at 55C and DNA was purified using DNA clean-up kit (Thermo Fisher Scientific). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 1.


title: OL152-ind-2

value: conditional chpT mutant


title: MC.PBS.PC#9

source_name: Mast Cell

value: Mast Cell


title: Topo-Seq, EcTopoI G116S M320V induced, replicate 2

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 2.

value: Exponential phase


title: OL152-ind-1


title: MC.PBS.PC#8


title: HCT116 - C-1 rep 1


title: Topo-Seq, EcTopoI G116S M320V induced, replicate 1

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 1.

value: DY330 topA-SPA


title: MC.PBS.PC#7


title: OL152-unind-2


title: OL152-unind-1


title: Control treated with RNAse HI, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 3

description: In exponentially growing culture CTD overexpression was induced from pCA25 CTD plasmid with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.

data_processing:
Library strategy: DRIP-Seq
Raw reads were trimmed and filtered using Trimmomatic (ILLUMINACLIP:2:30:10 LEADING:0 TRAILING:0 SLIDINGWINDOW:4:0 MINLEN:30).
Processed reads were aligned to the E. coli w3110 MuSGS genome with BWA MEM (default settings).
Strand-specific coverage for forward and reverse strands were obtained based on read alignments stored in SAM files using custom script (see https://github.com/sutormin94/E_coli_DRIP-Seq_analysis).
Genome_build: NC_007779.1_w3110_Mu (custom build, fasta and genome annotation are provided as supplementary files)
Supplementary_files_format_and_content: wig files with strand-specific sequencing coverage depth.

source_name: Escherichia coli DY330 topA-SPA pCA25 CTD

growth_protocol: For starter culture, a single colony of E. coli DY330 topA-SPA was inoculated in 5 mL of LB supplemented with 50 mkg/ml kanamycin and 0.5% glucose. The starter was grown overnight at 37C with shaking 180 rpm. 0.5 mL of the starter culture was inoculated in 50 mL of fresh LB supplemented with 0.5% glucose, and the culture was cultivated at 37C with shaking 180 rpm until reaching OD600=0.6. For cells expressing EcTopoI 14-kDa CTD cultivation was performed similarly but with E. coli DY330 topA-SPA harboring pCA24 14kDa CTD plasmid. Cultures were grown in LB supplemented with chloramphenicol 34 µg/ml and 0.5% glucose (for catabolyte repression).

extract_protocol:
Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. To shear nucleic acids, sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). As a control, an aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37oC and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot.
NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. DNA sequencing was performed using Illumina NextSeq with 75+75 bp paired-end protocol. Libraries preparation and sequencing were performed at Skoltech Sequencing Core Facilities. DRIP-Seq was performed in triplicate.

treatment_protocol: For the untreated sample, when OD600 reached 0.6, cells were harvested by centrifugation (4500 g, 5min, 4°C). For Rif sample, cell culture was pretreated with 100 µg/ml rifampicin for 20 minutes before cells harvesting. For CTD sample, a culture was cultivated until reaching OD600~0.2. Then, IPTG was added to final concentration 1 mM to induce EcTopoI 14-kDa CTD overexpression and the cultivation proceeded for 1 h before the culture harvesting (OD600~0.6). For CTD/Rif sample, CTD overexpression was induced with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes before cells harvesting.

value: pCA24 14kDa CTD

value: Overexpression of EcTopoI 14-kDa CTD was induced for 1 h with 1 mM IPTG and then treated with 100 mkg/ml rifampicin for 20 min (CTD/Rif)

value: Control treated with RNAse HI


title: B6_D_R2

description:
cancer derived cell line
B6_D_R2

value: vehicle treated

value: NFE2L1 KO2


title: Control treated with RNAse HI, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 2

description: In exponentially growing culture CTD overexpression was induced from pCA25 CTD plasmid with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2.


title: MC.PBS.PC#5

value: Peritoneal Cavity


title: WT-ind-1

source_name: WT-ind


title: MC.PBS.PC#6


title: DRIP-Seq, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 2

description: In exponentially growing culture CTD overexpression was induced from pCA25 CTD plasmid with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2.


title: MC.PBS.PC#3


title: WT-ind-2


title: DRIP-Seq, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 3

description: In exponentially growing culture CTD overexpression was induced from pCA25 CTD plasmid with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.


title: MC.PBS.PC#4


title: Topo-Seq, EcTopoI G116S M320V induced, replicate 3

description: Exponentially growing culture (OD600=0.4, 50 mL) in LB supplemented with chloramphenicol (34 µg/ml) and 0.5% glucose was induced with 10 mM arabinose. Cultivation proceeded for 30 min (final OD600~0.6) and cells were harvested by centrifugation (3000 g, 5min, 4C). Cells pellet was snap-frozen in liquid N2 and stored at -80C until further processing. The cell pellet was resuspended in 1 mL of Strap-Tactin lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl) containing protease inhibitors cocktail (1x concentration, cOmplete ULTRA, Sigma-Aldrich) and RNase A (0.1 mg/ml, Thermo Scientific). To disrupt cells and shear DNA, cells were sonicated in a 1.5 ml Eppendorf tube in an ice-water bath to achieve 100-1000 bp fragments range. Lysate was clarified by centrifugation at 8000 g for 5 min at 4C, and the resulting supernatant was used for further analysis. For cleavage complexes immunoprecipitation, 900 ul of the lysate was mixed with 80 μl of Strep-Tactin Superflow Plus affinity resin (Qiagen) pre-equilibrated with Strap-Tactin lysis Buffer supplemented with 0.05% SDS. After 1 h of incubation at room temperature with constant rotation the resin was washed three times with 1 mL of Strap-Tactin lysis Buffer, the proteins bound were eluted using 100 μl of Elution Buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 2.5 mM desthiobiotin). 80 µL of the elution were treated with Proteinase K (Thermo-Fisher Scientific) overnight at 50C. IP-DNA was purified using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 150+150 bp paired-end reads protocol. Biological replicate 3.

value: pBAD33 topA_strepII G116S M320V


title: MC.PBS.PC#2


title: WT-unind-1

source_name: WT-unind


title: DRIP-Seq, EcTopoI 14-kDa CTD overexpression and Rif 100 mkg/ml for 20 min, replicate 1

description: In exponentially growing culture CTD overexpression was induced from pCA25 CTD plasmid with 1mM IPTG at OD600~0.2 and the culture was cultivated for 1 h. Then, the culture was treated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 1.


title: WT-unind-2


title: Control treated with RNAse HI, Rif 100 mkg/ml for 20 min, replicate 3

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.

source_name: Escherichia coli DY330 topA-SPA

value: Treated with 100 mkg/ml rifampicin for 20 min (Rif)


title: MC.PBS.PC#1


title: Caput sperm DNA - H3K27ac_rep2

city: College Station

first: MICHAEL

middle: C.

last: GOLDING

state: Texas

address: 4466 TAMU

department: Physiology

institute: Texas A&M UNiversity

data_processing:
Basecalls performed using Sanger/Illumina version 1.9
ChIP-seq reads were trimmed for adaptors and quality and aligned to the mm10 genome assembly using BWA-MEM
BAM alignments were filtered on read mapping quality >=20
Peaks were called using MACS2 version 2.1.1.20160309.6 with an extension size of 150 and FDR 0.05.
Genome_build: mm10
Supplementary_files_format_and_content: wig files were generated using the Wig/BedGraph-to-bigWig converter tool

source_name: Caput spermatozoa

growth_protocol: We obtained postnatal day 90 adult C57BL/6N (RRID:IMSR_JAX:005304) males from the Texas A&M Institute for Genomic Medicine. We maintained males on a standard diet (catalog# 2019, Teklad Diets, Madison, WI, USA) and 12-hour light/dark cycle.

extract_protocol:
Sperm histone ChIP was carried out using a previously published protocol (Hisano et al., 2013). Briefly, for each individual biological rep, sperm collected from the Caputs and Cauda of 10-15 mice were pooled, purified and pre-treated with 50μM DTT for 2h at RT to open sperm chromatin. Cell suspensions were split into roughly 15-20 million sperm aliquots per IP and treated with 90 U of MNase (New England Biolabs, #M0247S) for 5 min at 37C. MNase-digested chromatin was then pre-cleared with blocked A/G Sepharose beads for 1h at 4C. For IP, 5 μg of antibody (H3K27ac or H3K9me2) was added to the pre-cleared chromatin overnight followed by 50 μL of blocked beads for 4 h at 4C. Beads were washed and DNA was then eluted in elution buffer. Following RNase A and proteinase K treatment, DNA was purified using phenol/chloroform isolation and ethanol precipitation and resuspended in 40 μL ultrapure water.
Library preparation using 10ng of DNA and 75 bp paired-end sequencing with at least 50 million reads per samples was carried out at the UT Health Cancer Genomics Center, Houston.

tag: region

value: Caput

value: spermatozoa

tag: chip antibody

value: H3K27ac (abcam, ab4729)

value: untreated


title: Control treated with RNAse HI, Rif 100 mkg/ml for 20 min, replicate 2

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2.


title: MOLM13_Selinexor_rep3

city: Durham

first: Kris

middle: C

last: Wood

state: NC

address: 450 Research Drive, LSRC C259, PO Box 3813

department: Department of Pharmacology and Cancer Biology

institute: Duke University

data_processing:
Alignment was performed using STAR alignment on R version 3.6.1
Differnential expression analysis was performed using DESeq2 R version 3.6.1
Genome_build: hg38
Supplementary_files_format_and_content: Processed files include raw counts, normalized counts and differential expression analysis between selinexor and DMSO treated cell lines

source_name: MOLM-13 cell line

growth_protocol: Cells were cultured in RPMI supplemented with 10% FBS and 1% pen-strep and grown at 37C with 5% CO2

extract_protocol:
Qiagen RNEasy Mini Kit
cDNA library, PolyA selected, non-strand specific

treatment_protocol: Cells were treated for 36 hours with indicated treatment

value: Leukemic blasts

tag: disease

value: AML

value: Selinexor (75nM) 36hours


title: MOLM13_Selinexor_rep2


title: Cauda sperm DNA - H3K27ac_rep2

source_name: Cauda spermatozoa

value: Cauda


title: Control treated with RNAse HI, Rif 100 mkg/ml for 20 min, replicate 1

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 1.

value: DY330 topA-SPA

value: Exponential phase


title: MC.IL9.PC#3

value: Mast Cell


title: MOLM13_Selinexor_rep1


title: MC.IL9.PC#2


title: Cauda sperm DNA - H3K27ac_rep1


title: Caput sperm DNA - H3K27ac_rep1


title: MC.IL9.PC#1


title: DRIP-Seq, Rif 100 mkg/ml for 20 min, replicate 2

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when it was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2.

value: IP


title: DRIP-Seq, Rif 100 mkg/ml for 20 min, replicate 3

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when it was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.


title: MOLM13_DMSO_rep2

value: DMSO


title: MOLM13_DMSO_rep3


title: DRIP-Seq, Rif 100 mkg/ml for 20 min, replicate 1

description: Exponentially growing in LB medium culture (OD600=0.6) was pretreated with 100 µg/ml rifampicin for 20 minutes when it was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 1.


title: Cauda sperm DNA - H3K9me2_rep2

value: H3K9me2 (Active Motif, 39240)


title: MC.IL4.PC#2

value: Peritoneal Cavity


title: Control treated with RNAse HI, EcTopoI 14-kDa CTD overexpression, replicate 3

description: Exponentially growing culture was cultivated until reaching OD600~0.2. Then, IPTG was added to final concentration 1 mM to induce EcTopoI 14-kDa CTD overexpression form the pCA25 CTD plasmid and the cultivation proceeded for 1 h when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.

value: pCA24 14kDa CTD

value: Overexpression of EcTopoI 14-kDa CTD was induced for 1 h with 1 mM IPTG (CTD)

value: Control treated with RNAse HI


title: Caput sperm DNA - H3K9me2_rep2

value: Caput

value: spermatozoa


title: OCIAML2_Selinexor_rep3

source_name: OCIAML2 cell line

value: Selinexor (200nM) 36hours


title: OCIAML2_Selinexor_rep2

value: Leukemic blasts


title: MC.IL21.PC#3


title: OCIAML2_Selinexor_rep1

value: AML


title: MC.IL4.PC#1


title: OCIAML2_DMSO_rep3


title: DKO_9

city: Gainesville

first: Eric

middle: T

state: FL

address: 2033 Mowry Rd

institute: University of Florida

data_processing:
Illumina bcl2fastq2 Conversion Software v2.20 for basecalling and demultiplexing
Pseudoalignment and Transcripts per Million (TPM) were calculated with Kallisto v0.44.0 to mm10 with parameters quant -b 100
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text file including TPM values for each Sample

source_name: MBNL1/2 DKO heart

extract_protocol:
Heart tissues were homogenized in Tri-reagent and RNA was precipitated using isopropanol. RNA pellets were washed with 75% ethanol and then dissolved in RNA storage solution.
Strand-specific paired-end RNA sequencing of total RNA was performed using the NEBNext Ultra II Directional Library Prep Kit.

tag: genotype/variation

value: MBNL1/2 DKO

value: heart


title: DKO_8


title: DRIP-Seq, EcTopoI 14-kDa CTD overexpression, replicate 3

description: Exponentially growing culture was cultivated until reaching OD600~0.2. Then, IPTG was added to final concentration 1 mM to induce EcTopoI 14-kDa CTD overexpression form the pCA25 CTD plasmid and the cultivation proceeded for 1 h when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. 10 µg of S9.6 antibodies (Kerafast, ENH001) were incubated with 20 µL of protein-G Sepharose (Bialexa) pre-equilibrated with PBS in 70 µL of IP-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 140 mM NaCl) for 2 h at 4C with gentle shaking. After the pre-incubation, an aliquot (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was added and the immunoprecipitation was continued for 4 h. Then, the resin was washed with 1 mL of IP-buffer, 1 mL of IPS-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 500 mM NaCl) and twice with 1 mL of Wash-buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton X-100, 250 mM NaCl). TES-buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 250 mM NaCl) was mixed with the washed resin to get 200 µL of slurry and proteinase K (ThermoFisher) was added to a final concentration 0.5 mg/mL. Proteolysis was performed at 55C for 1 h. After that, the resin was spun down by centrifugation and nucleic acids were purified from the supernatant using AMPure XP magnetic beads (Beckman Coulter). NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.

value: DY330 topA-SPA

tag: treatment


title: MC.IL21.PC#2


title: MC.IL21.PC#1


title: OCIAML2_DMSO_rep2


title: DKO_7

value: WT


title: WT_6

source_name: WT mouse heart


title: MOLM13_DMSO_rep1


title: MC.IL4.PC#3

value: Mast Cell


title: OCIAML2_DMSO_rep1


title: Control treated with RNAse HI, no treatment, replicate 3

description: Exponentially growing culture (OD600=0.6) in LB was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 3.

tag: growth phase

value: Exponential phase

value: no treatment


title: MC.IL2.PC#2


title: Caput sperm DNA - H3K9me2_rep1

value: H3K9me2 (Active Motif, 39240)

value: untreated


title: Control treated with RNAse HI, EcTopoI 14-kDa CTD overexpression, replicate 2

description: Exponentially growing culture was cultivated until reaching OD600~0.2. Then, IPTG was added to final concentration 1 mM to induce EcTopoI 14-kDa CTD overexpression form the pCA25 CTD plasmid and the cultivation proceeded for 1 h when the culture was harvested by centrifugation (4500 g, 5min, 4°C). Total nucleic acids were purified from cells pellet using GeneJET Genomic DNA purification kit (ThermoFisher) according to the manufacturer's protocol but omitting the RNAse A treatment step. Sample was sonicated using Covaris ultrasonicator to get median fragment size 150 bp. An aliquot of sonicated nucleic acids (containing 6 µg of sheared DNA, concentrations measured using Qubit 1X dsDNA HS Assay) was treated with 5U of RNAse HI (NEB) in RNAse HI buffer for 30 min at 37C and then nucleic acids were purified with GeneJET Gel Extraction and DNA cleanup micro kit (General cleanup protocol, ThermoFisher). IP for treated aliquot using S9.6 antibodies and nucleic acid purification were performed as described for the untreated aliquot. NGS libraries were prepared using strand-specific Accel NGS 1S kit (Swift Bioscience) suitable for single-stranded DNA. Sequencing was performed with Illumina NextSeq with 75+75 bp paired-end reads protocol. Biological replicate 2.

value: pCA24 14kDa CTD

tag: optical density at 600 nm