title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S47_CGATGT


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S47_CTTGTA

status: Public on Jul 13 2023

type: SRA

city: Philadelphia

first: Hao

last: Wu

state: PA

address: 415 Curie Boulevard

department: Genetics

country: USA

institute: University of Pennsylvania

library_source: genomic

data_processing:
The pre-processing (read alignment, quality filtering and read deduplication) was performed for snhmC/Joint-snhmC-seq datasets of individual single cells as previously described with minor modifications6. Briefly, demultiplexing of inline barcodes was first performed allowing up to 1-nt mismatch. The data quality was examined with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Raw sequencing reads were trimmed for adaptor sequences and inline barcodes using Cutadapt with the following parameters in paired-end mode: -f fastq -q 20 -u 16 -U 16 -m 30 -a AGATCGGAAGAGCACACGTCTGAAC -A AGATCGGAAGAGCGTCGTGTAGGGA. The trimmed R1 and R2 reads were mapped independently against the reference genome (mm10) using Bismark (v0.18.2) with following parameters: --bowtie2 -D 15 -R 2 -L 20 -N 0 –score_min L,0,-0.2 (--pbat option was turned on for mapping R1 reads). Uniquely mapped reads were filtered for minimal mapping quality (MAPQ>=10) using samtools9. PCR duplicates were removed using the Picard MarkDuplicates (http://broadinstitute.github.io/picard/). To eliminate reads from strands not deaminated by A3A, reads with three or more consecutive non-converted cytosines in the CH context were removed using filter_non_conversion in Bismark. Low quality snhmC/Joint-snhmC-seq datasets were removed using the following set of criteria. First, Mapping rate of raw sequencing reads is set to be at least 20%; Second, we set a minimum on the number of pass filter reads (uniquely mapped, deduplicated, MAPQ>=10, <=3 consecutive 5mCH sites) to be 100,000 per cell; Third, the 5mCG deamination rate in spike-in lambda phage control is higher than 90%. Base calling of unmethylated and methylated cytosines was performed by bismark_methylation_extractor in Bismark in each individual nucleus. 5hmC signals were calculated as % of C/(C+T) at each cytosine base.
Assembly: mm10
Supplementary files format and content: Tab-delimited bismark methylation calling files include genomic coordinates (columan 1-3: chromosome, start, and end), 5hmC signal (column 4: % of 5hmC levels normalized to total reads), 5hmC read (column 5), and unmodified read (column 6) for all detected CpG (._CpG.mm10.bismark.cov)  or CpH (._CpH.mm10.bismark.cov) sites across the mouse genome in individually sequenced cell.
Library strategy: bisulfite-assisted ACE-seq

molecule: genomic DNA

organism: Mus musculus

source_name: Cortex

growth_protocol: All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. Mice were group-housed in cages of three to five in a 12-hr light/dark cycle with food and water provided ad libitum. All mice used for experiments were naïve to behavioral assays and other procedures. For all bulk-nucleus and single-nucleus experiments, 6-10 weeks old, wild-type, male mice in a pure C57BL/6J background were used. For prospectively isolating inhibitory (Inh) and excitatory (Ex) neuronal subtypes to benchmark snhmC-seq and joint-snhmC-seq, 9-week-old transgenic mice (Mecp2Tavi/y, NexCre/+, Rosa26BirA/BirA) from a C57BL/6J background were used as previously described (Johnson et al. 2017; PMID: 28920956).

extract_protocol:
Nuclei isolation from mouse cortical tissues was performed as described with minor modifications (Hu* & Fabyanic* et al., 2017; PMID: 29220646). Briefly, mouse brains were rapidly resected on ice. Cortices were flash-frozen in liquid nitrogen and stored at -80°C before nuclear isolation. Nuclei were isolated and purified as previously described with some modifications3. Briefly, 14mL sucrose cushion (1.8M sucrose (Sigma-Aldrich, RNase and DNase free, ultra-pure grade), 10mM Tris-HCl pH 8.0, 3mM MgAc2 (Sigma-Aldrich), and protease inhibitor cocktail (Roche)) was added to the bottom of centrifuge tubes (Beckman Coulter). Using a glass homogenizer (Wheaton), a frozen mouse cortical sample was dounce homogenized (19 times with loose pestle followed by 4 times with tight pestle) in 12mL homogenization buffer (0.32M sucrose, 5mM CaCl2 (Sigma-Aldrich), 3mM MgAc2, 10mM Tris-HCl pH 8.0, 0.1% Triton X-100 (Sigma-Aldrich), 0.1mM EDTA (Invitrogen), protease inhibitor cocktail (Roche)). Homogenates (~12ml) were carefully layered onto the sucrose cushion in the centrifuge tubes, and 10mL homogenization buffer was added atop of the homogenates. The tubes were then balanced and centrifuged in a Beckman Coulter L7-65 Ultracentrifuge at 82,705 g at 4°C for 2hr using a Beckman Coulter SW28 swing bucket rotor (Beckman Coulter). The supernatant was carefully removed via aspiration. 1ml chilled DPBS was added to resuspend the nuclear pellet, and nuclei were subsequently transferred to a 1.5-mL tube. Nuclei were pelleted at 500 g for 10 min at 4C, and then resuspended in 0.01% BSA (Sigma-Aldrich) in DPBS. After resuspension, nuclei were filtered through a 40-μm cell strainer (Fisher Scientific), visually inspected for morphology and quality assurance, and counted using a Fuchs-Rosenthal counting chamber before FANS.
All steps of library preparation were performed in a horizontal laminar flow hood (AirCleanⓇ600 PCR Workstation) to minimize environmental DNA contamination. To profile both 5hmC and true 5mC in the same single cell through physical splitting, bisulfite conversion of single nuclei was first carried out using the EZ-96 DNA Methylation-Direct™ Kit (Zymo, Deep Well Format, D5023). Briefly, 96-well plates containing FANS-isolated single nuclei were heated at 50°C for 20min and stored in -80°C for later use. 32.5μL CT Conversion Reagent was added to each well, followed by sealing the plate and shaking to mix. Plates were treated with the following program using a thermocycler: two cycles of [98°C for 8min, 64°C for 1h45min], and 4°C forever. Purification was performed using Zymo-Spin™ I-96 Binding Plates preloaded with 250μL M-binding buffer according to kit instructions with following modifications. For the Joint-snhmC-seq, 20µl of nuclease-free water was used as the elution buffer. The 9µl eluent retained for the snmC-seq2/split (bisulfite-only) workflow will proceed directly to random priming. To the other 9μl eluent retained for snhmC-seq2/split workflow, 7µl nuclease-free water was added to the eluant, for a total reaction volume of 16µl. The 16µl of bisulfite-treated DNA were denatured at 95°C for 1min and snap cooled by transfer to an isopropanol dry ice bath and then to the −80°C pre-incubated PCR tube rack. To snap-cooled samples, 8µl of deamination enzyme mix (2.4µl of 10x reaction buffer (50mM Bis-Tris (pH 6.0) with 0.1% Triton X-100), 0.48µl BSA, 0.48µl of commercial A3A (at a final concentration of ~500 nM, NEB, E7125L) or in-house A3A (~1000 nM), and 4.64µL nuclease-free water) was added into each well. The deamination reactions were incubated for 30 minutes at 37°C, tapped to mix, and spun down before undergoing an additional incubation for 2.5h at 37°C. Post-incubation, these reactions were purified with 2x commercial SPRI beads (Beckman Coulter B23318), and eluted in 9µl of Low EDTA TE buffer (IDT). For the Joint-snhmC-seq, P5L random primers were added to split single nuclei samples as described for snhmC-seq. Briefly, 1µl of matched, individual 250µM random primers was added to each reaction (9µl) to allow for subsequent multiplexing (up to 24-plex) and pairing of snmC-seq2/split to snhmC-seq2/split reactions of the same single cell. Plates were then heated at 95°C using a thermocycler for 3min to denature and were immediately chilled on ice for 2min. Next, 10µl of enzyme mix (2µl o f10x Isothermal Amplification Buffer II, 0.8µl of Bst 3.0 DNA Polymerase (8,000 U/ml), 1.2µl 100 mM MgSO4 (these three reagents all from NEB M0374L), 2.8µl of 10mM dNTP (NEB N0447L), and 3.2µl water) was added to each well (a 20µl reaction) and reactions were mixed by shaking. Reactions were then incubated at 60°C for 1h. Following this, 2μL Exonuclease 1 (20U/μl, Enzymatics X8010L) and 1μL Shrimp Alkaline Phosphatase (rSAP) (1U/μl, NEB M0371L) was added to each reaction followed by vortexing and incubation in a thermocycler at 37°C for 30min followed by 4°C forever. To multiplex subsequent adaptor ligation reactions, 18.4μl (0.8x) homebrew SPRI beads were added to each reaction and 8 sample/bead mixtures were pooled (up to 24 samples if all inline barcodes were used). The sample/bead mixtures were incubated at room temperature for 5min before being placed on a 96-well magnetic separator (DynaMag™-96 Side Magnet, ThermoFisher 12331D). Supernatant was removed from each well, followed by three rounds of washing with 180μl 80% ethanol. Beads were air dried at room temperature and 10μl Low EDTA TE buffer (IDT) was added to each well to fully resuspend the beads. Eluted samples were then transferred to either new PCR strips (for bulk sample) or 96-well plates (for single nuclei). The reactions were denatured in a thermocycler at 95°C for 3min and subsequently chilled on ice for 2min. 10.5μl Adaptase master mix (2μl Buffer G1, 2μl Reagent G2, 1.25μl Reagent G3, 0.5μl Enzyme G4, 0.5μl Enzyme G5, and 4.25μl Low EDTA TE buffer from xGenTM Methyl-Seq Lib Prep kit, IDT, 10009860) was added to each reaction, followed by vortexing. Reactions were incubated in a thermocycler at 37°C for 30min then 4°C forever. Finally, to amplify the pooled library and add dual indexing barcodes, 30μl PCR mix (25μl KAPA HiFi HotStart ReadyMix, Roche/KAPA Biosystems KK2602, 1μl of 30μM P5 (D501-508) indexing primer, and 5μl of 10μM P7 (D701-712) indexing primer) were added to each well, followed by mixing with vortexing. Reactions were transferred to a thermocycler programmed with the following stages: 95°C for 2min, 98°C for 30sec, 15 cycles of [98°C for 15sec, 64°C for 30sec, 72°C for 2min] (for pooled single nucleus reactions), 72°C for 5min, and 4°C forever. For bulk samples, we perform qPCR and subtract two cycles from the inflection point for each sample to determine optimal cycle number for amplification. PCR products were cleaned with two rounds of 0.8x SPRI beads, concentration was determined via QbitⓇ dsDNA High Sensitivity Assay Kit (Invitrogen Q32851), and library size was determined via Bioanalyzer (Agilent High Sensitivity DNA Kit, 5067-4626). Reactions were sequenced on Illumina NovaSeq 6000 (300-cycle v1.5 kit) sequencers.

tag: tissue

value: Cortex

tag: developmental stage

value: Postnatal 6-10 weeks

tag: Sex

value: Male

tag: genotype

value: wild-type

tag: treatment


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S47_ACTTGA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_TGACCA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S47_ATCACG


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_GCCAAT


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_CTTGTA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_CGATGT


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_CAGATC


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_ACTTGA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_TGACCA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_ATCACG


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_GCCAAT


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_CGATGT


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_CTTGTA


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_CAGATC


title: 7E_M_1791

city: La Jolla

first: Joseph

middle: R

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

data_processing:
Reads trimed with Cutadapt for adapters and low quality bases (Phred score >= 20)
The resulting files are mapped to in-silico bisulfite converted mm10 reference genome using Bismark.
The raw BAM file will be filtered using samtools to filter reads by MAPQ > 10 and use Picard to remove PCR duplicates
Tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls will be generated with in-house developing package using the filtered BAM files
Assembly: mm10
Supplementary files format and content: tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls. Columns are: 1. Chromosome, 2. Position, 3. Strand, 4. Cytosin Context, 5. methylated base call, 6. total mapped base call, 7. not used, place holder column
Library strategy: snmC-seq2

source_name: C57BL/6J Male P56 Mouse Brain

growth_protocol: All mice are C57BL/6J Male, brain dissected in P56

extract_protocol:
the brain of P56 male C57BL/6J mouse will be removed and cut into 600 microns thick coronal sections using a tissue matrix. Then each region will be microdissected from the coronal tissue sections based on identified landmarks and brain atlas coordinates, as defined in Allen brain atlas CCF3 (Avaliable at: http://atlas.brain-map.org). For each dissection region, the tissue from several littermate mice will be combined together into to separate replicate groups. The number of animals in a replicate depends on the size of the tissue. Nuclei preparation will be stained with the fluorescently labeled anti-NeuN body and nucleic acid stain Hoechst 33342.
Library prepared following protocol discribed in Luo, Chongyuan, Angeline Rivkin, Jingtian Zhou, Justin P. Sandoval, Laurie Kurihara, Jacinta Lucero, Rosa Castanon, et al. 2018. “Robust Single-Cell DNA Methylome Profiling with snmC-seq2.” Nature Communications 9 (1): 3824.

tag: brain region

tag: age

tag: strain


title: 7E_M_1789


title: 7E_M_1788


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S48_ACTTGA


title: 7E_M_1721


title: 7E_M_1723


title: 7E_M_1722


title: 7E_M_1719


title: 7E_M_1717


title: 7E_M_1718


title: 7E_M_1720


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S46_TAGCTT


title: 7E_M_1714


title: 7E_M_1715


title: 7E_M_1716


title: 7E_M_1712


title: snhmCseq2.split_Cortex.WT_NeuN_pos_novaseq08.03.2021_S45_TAGCTT


title: 7E_M_1713


title: 7E_M_1711


title: 7E_M_1710


title: 7E_M_1708


title: 7E_M_1707


title: 7E_M_1706


title: 7E_M_1790


title: 7E_M_1705


title: 7E_M_1700


title: 7E_M_1701


title: 7E_M_1703


title: 7E_M_1709


title: 7E_M_1702


title: 7E_M_1704


title: 7E_M_1699


title: 7E_M_1697


title: 7E_M_1695


title: 7E_M_1696


title: 7E_M_1694


title: 7E_M_1693


title: 7E_M_1691


title: 7E_M_1698


title: 7E_M_1692


title: 7E_M_1690


title: 7E_M_1688


title: 7E_M_1687


title: 7E_M_1689


title: 7E_M_1686


title: 7E_M_1684


title: 7E_M_1685


title: 7E_M_1682


title: 7E_M_1683


title: 7E_M_1679


title: 7E_M_1678


title: 7E_M_1681


title: 7E_M_1680


title: 7E_M_1677


title: 7E_M_1676


title: 7E_M_1675


title: 7E_M_1672


title: 7E_M_1674


title: 7E_M_1669


title: 7E_M_1667


title: 7E_M_1673


title: 7E_M_1671


title: 7E_M_1670


title: 7E_M_1668


title: 7E_M_1665


title: 7E_M_1663


title: 7E_M_1664


title: 7E_M_1662


title: 7E_M_1661


title: 7E_M_1403


title: 7E_M_1402


title: 7E_M_1401


title: 7E_M_1399


title: 7E_M_1398


title: 7E_M_1660


title: 7E_M_1400


title: 7E_M_1397


title: 7E_M_1394


title: 7E_M_1395


title: 7E_M_1392


title: 7E_M_1396


title: 7E_M_1391


title: 7E_M_1389


title: 7E_M_1390


title: 7E_M_1666


title: 7E_M_1388


title: 7E_M_1387


title: 7E_M_1384


title: 7E_M_1381


title: 7E_M_1379


title: 7E_M_1383


title: 7E_M_1385


title: 7E_M_1378


title: 7E_M_1386


title: 7E_M_1377


title: 7E_M_1382


title: 7E_M_1375


title: 7E_M_1372


title: 7E_M_1370


title: 7E_M_1393


title: 7E_M_1376


title: 7E_M_1368


title: 7E_M_1371


title: 7E_M_1373


title: 7E_M_1363


title: 7E_M_1362


title: 7E_M_1367


title: 7E_M_1364


title: 7E_M_1360


title: 7E_M_1374


title: 7E_M_1380


title: 7E_M_1366


title: 7E_M_1361


title: 7E_M_1359


title: 7E_M_1357


title: 7E_M_1369


title: 7E_M_1356


title: 7E_M_1355


title: 7E_M_1350


title: 7E_M_1351


title: 7E_M_1352


title: 7E_M_1348


title: 7E_M_1349


title: 7E_M_1354


title: 7E_M_1365


title: 7E_M_1343


title: 7E_M_1344


title: 7E_M_1347


title: 7E_M_1345


title: 7E_M_1346


title: 7E_M_1341


title: 7E_M_1340


title: 7E_M_1342


title: 7E_M_1338


title: 7E_M_1339


title: 7E_M_1336


title: 7E_M_1334


title: 7E_M_1335


title: 7E_M_1353


title: 7E_M_1358


title: 7E_M_1333


title: 7E_M_1332


title: 7E_M_1331


title: 7E_M_1329


title: 7E_M_1327


title: 7E_M_1326


title: 7E_M_1325


title: 7E_M_1328


title: 7E_M_1330


title: 7E_M_1320


title: 7E_M_1322


title: 7E_M_1319


title: 7E_M_1316


title: 7E_M_1315


title: 7E_M_1312


title: 7E_M_1337


title: 7E_M_1310


title: 7E_M_1311


title: 7E_M_1308


title: 7E_M_1306


title: 7E_M_1304


title: 7E_M_1307


title: 7E_M_1323


title: 7E_M_1303


title: 7E_M_1305


title: 7E_M_1301


title: 7E_M_1300


title: 7E_M_1321


title: 7E_M_1299


title: 7E_M_1297


title: 7E_M_1296


title: 7E_M_1294


title: 7E_M_1318


title: 7E_M_1295


title: 7E_M_1291


title: 7E_M_1293


title: 7E_M_1309


title: 7E_M_1292


title: 7E_M_1313


title: 7E_M_1290


title: 7E_M_1289


title: 7E_M_1317


title: 7E_M_1288


title: 7E_M_1285


title: 7E_M_1287


title: 7E_M_1302


title: 7E_M_1298


title: 7E_M_1286


title: 7E_M_1283


title: 7E_M_1280


title: 7E_M_1278


title: 7E_M_1282


title: 7E_M_1146


title: 7E_M_1281


title: 7E_M_1279


title: 7E_M_1277


title: 7E_M_1147


title: 7E_M_1144


title: 7E_M_1145


title: 7E_M_1141


title: 7E_M_1143


title: 7E_M_1142


title: 7E_M_1284


title: 7E_M_1140


title: 7E_M_1138


title: 7E_M_1135


title: 7E_M_1136


title: 7E_M_1139


title: 7E_M_1131


title: 7E_M_1129


title: 7E_M_1132


title: 7E_M_1133


title: 7E_M_1128


title: 7E_M_1134


title: 7E_M_1276


title: 7E_M_1126


title: 7E_M_1130


title: 7E_M_1125


title: 7E_M_1124


title: 7E_M_1137


title: 7E_M_1123


title: 7E_M_1122


title: 7E_M_1119


title: 7E_M_1115


title: 7E_M_1114


title: 7E_M_1117


title: 7E_M_1111


title: 7E_M_1112


title: 7E_M_1118


title: 7E_M_1120


title: 7E_M_1121


title: 7E_M_1113


title: 7E_M_1127


title: 7E_M_1110


title: 7E_M_1108


title: 7E_M_1105


title: 7E_M_1103


title: 7E_M_1106


title: 7E_M_1104


title: 7E_M_1100


title: 7E_M_1101


title: 7E_M_1109


title: 7E_M_1099


title: 7E_M_1102


title: 7E_M_1098


title: 7E_M_1097


title: 7E_M_1116


title: 7E_M_1093


title: 7E_M_1095


title: 7E_M_1107


title: 7E_M_1091


title: 7E_M_1089


title: 7E_M_1086


title: 7E_M_1088


title: 7E_M_1084


title: 7E_M_1018


title: 7E_M_1087


title: 7E_M_1016


title: 7E_M_1085


title: 7E_M_1015


title: 7E_M_1008


title: 7E_M_1009


title: 7E_M_1010


title: 7E_M_1013


title: 7E_M_1011


title: 7E_M_1096


title: 7E_M_1012


title: 7E_M_1014


title: 7E_M_1017


title: 7E_M_1007


title: 7E_M_1004


title: 7E_M_1003


title: 7E_M_1094


title: 7E_M_1019


title: 7E_M_1006


title: 7E_M_1002


title: 7E_M_1001


title: 7E_M_1090


title: 7E_M_1092


title: 7E_M_1000


title: 7E_M_995


title: 7E_M_994


title: 7E_M_997


title: 7E_M_996


title: 7E_M_999


title: 7E_M_993


title: 7E_M_992


title: 7E_M_991


title: 7E_M_990


title: 7E_M_988


title: 7E_M_989


title: 7E_M_986


title: 7E_M_984


title: 7E_M_981


title: 7E_M_983


title: 7E_M_982


title: 7E_M_985


title: 7E_M_987


title: 7E_M_980


title: 7E_M_978


title: 7E_M_976


title: 7E_M_975


title: 7E_M_973


title: 7E_M_972


title: 7E_M_974


title: 7E_M_971


title: 7E_M_979


title: 7E_M_970


title: 7E_M_998


title: 7E_M_969


title: 7E_M_967


title: 7E_M_966


title: 7E_M_968


title: 7E_M_965


title: 7E_M_963


title: 7E_M_962


title: 7E_M_961


title: 7E_M_960


title: 7E_M_958


title: 7E_M_636


title: 7E_M_632


title: 7E_M_637


title: 7E_M_634


title: 7E_M_633


title: 7E_M_631


title: 7E_M_627


title: 7E_M_626


title: 7E_M_624


title: 7E_M_630


title: 7E_M_625


title: 7E_M_620


title: 7E_M_623


title: 7E_M_628


title: 7E_M_629


title: 7E_M_622


title: 7E_M_621


title: 7E_M_619


title: 7E_M_959


title: 7E_M_964


title: 7E_M_613


title: 7E_M_617


title: 7E_M_618


title: 7E_M_611


title: 7E_M_616


title: 7E_M_615


title: 7E_M_614


title: 7E_M_957


title: 7E_M_607


title: 7E_M_608


title: 7E_M_635


title: 7E_M_609


title: 7E_M_604


title: 7E_M_606


title: 7E_M_603


title: 7E_M_600


title: 7E_M_597


title: 7E_M_599


title: 7E_M_596


title: 7E_M_602


title: 7E_M_598


title: 7E_M_594


title: 7E_M_605


title: 7E_M_601


title: 7E_M_593


title: 7E_M_595


title: 7E_M_592


title: 7E_M_590


title: 7E_M_591


title: 7E_M_589


title: 7E_M_587


title: 7E_M_585


title: 7E_M_584


title: 7E_M_582


title: 7E_M_586


title: 7E_M_583


title: 7E_M_580


title: 7E_M_588


title: 7E_M_581


title: 7E_M_579


title: 7E_M_577


title: 7E_M_578


title: 7E_M_576


title: 7E_M_575


title: 7E_M_574


title: 7E_M_567


title: 7E_M_569


title: 7E_M_572


title: 7E_M_571


title: 7E_M_568


title: 7E_M_570


title: 7E_M_573


title: 7E_M_565


title: 7E_M_564


title: 7E_M_563


title: 7E_M_562


title: 7E_M_561


title: 7E_M_566


title: 7E_M_560


title: 7E_M_559


title: 7E_M_555


title: 7E_M_556


title: 7E_M_557


title: 7E_M_553


title: 7E_M_554


title: 7E_M_551


title: 7E_M_550


title: 7E_M_549


title: 7E_M_547


title: 7E_M_546


title: 7E_M_545


title: 7E_M_544


title: 7E_M_541


title: 7E_M_539


title: 7E_M_543


title: 7E_M_534


title: 7E_M_535


title: 7E_M_537


title: 7E_M_558


title: 7E_M_533


title: 7E_M_538


title: 7E_M_531


title: 7E_M_540


title: 7E_M_530


title: 7E_M_536


title: 7E_M_529


title: 7E_M_552


title: 7E_M_548


title: 7E_M_524


title: 7E_M_525


title: 7E_M_532


title: 7E_M_526


title: 7E_M_528


title: 7E_M_523


title: 7E_M_522


title: 7E_M_520


title: 7E_M_519


title: 7E_M_518


title: 7E_M_521


title: 7E_M_542


title: 7E_M_517


title: 7E_M_516


title: 7E_M_515


title: 7E_M_514


title: 7E_M_513


title: 7E_M_382


title: 7E_M_512


title: 7E_M_511


title: 7E_M_527


title: 7E_M_379


title: 7E_M_378


title: 7E_M_380


title: 7E_M_381


title: 7E_M_377


title: 7E_M_371


title: 7E_M_375


title: 7E_M_372


title: 7E_M_376


title: 7E_M_373


title: 7E_M_374


title: 7E_M_369


title: 7E_M_370


title: 7E_M_368


title: 7E_M_366


title: 7E_M_364


title: 7E_M_365


title: 7E_M_361


title: 7E_M_362


title: 7E_M_363


title: 7E_M_360


title: 7E_M_359


title: 7E_M_356


title: 7E_M_354


title: 7E_M_355


title: 7E_M_353


title: 7E_M_352


title: 7E_M_351


title: 7E_M_350


title: 7E_M_347


title: 7E_M_348


title: 7E_M_367


title: 7E_M_343


title: 7E_M_345


title: 7E_M_341


title: 7E_M_342


title: 7E_M_337


title: 7E_M_339


title: 7E_M_336


title: 7E_M_346


title: 7E_M_332


title: 7E_M_357


title: 7E_M_334


title: 7E_M_330


title: 7E_M_358


title: 7E_M_335


title: 7E_M_349


title: 7E_M_327


title: 7E_M_328


title: 7E_M_325


title: 7E_M_326


title: 7E_M_323


title: 7E_M_344


title: 7E_M_322


title: 7E_M_340


title: 7E_M_338


title: 7E_M_321


title: 7E_M_319


title: 7E_M_320


title: 7E_M_318


title: 7E_M_333


title: 7E_M_316


title: 7E_M_317


title: 7E_M_331


title: 7E_M_314


title: 7E_M_315


title: 7E_M_310


title: 7E_M_309


title: 7E_M_311


title: 7E_M_329


title: 7E_M_308


title: 7E_M_306


title: 7E_M_307


title: 7E_M_305


title: 7E_M_303


title: 7E_M_304


title: 7E_M_301


title: 7E_M_324


title: 7E_M_302


title: 7E_M_299


title: 7E_M_300


title: 7E_M_297


title: 7E_M_296


title: 7E_M_295


title: 7E_M_313


title: 7E_M_293


title: 7E_M_292


title: 7E_M_287


title: 7E_M_285


title: 7E_M_284


title: 7E_M_290


title: 7E_M_282


title: 7E_M_312


title: 7E_M_288


title: 7E_M_286


title: 7E_M_283


title: 7E_M_289


title: 7E_M_278


title: 7E_M_281


title: 7E_M_277


title: 7E_M_298


title: 7E_M_279


title: 7E_M_276


title: 7E_M_294


title: 7E_M_275


title: 7E_M_269


title: 7E_M_271


title: 7E_M_272


title: 7E_M_274


title: 7E_M_268


title: 7E_M_273


title: 7E_M_267


title: 7E_M_266


title: 7E_M_265


title: 7E_M_264


title: 7E_M_263


title: 7E_M_261


title: 7E_M_262


title: 7E_M_260


title: 7E_M_280


title: 7E_M_257


title: 7E_M_256


title: 7E_M_258


title: 7E_M_255


title: 7E_M_124


title: 7E_M_125


title: 7E_M_123


title: 7E_M_270


title: 7E_M_118


title: 7E_M_117


title: 7E_M_122


title: 7E_M_119


title: 7E_M_109


title: 7E_M_115


title: 7E_M_107


title: 7E_M_112


title: 7E_M_113


title: 7E_M_259


title: 7E_M_116


title: 7E_M_111


title: 7E_M_108


title: 7E_M_121


title: 7E_M_104


title: 7E_M_103


title: 7E_M_105


title: 7E_M_102


title: 7E_M_101


title: 7E_M_99


title: 7E_M_106


title: 7E_M_100


title: 7E_M_114


title: 7E_M_98


title: 7E_M_95


title: 7E_M_97


title: 7E_M_96


title: 7E_M_120


title: 7E_M_94


title: 7E_M_91


title: 7E_M_92


title: 7E_M_93


title: 7E_M_90


title: 7E_M_89


title: 7E_M_84


title: 7E_M_81


title: 7E_M_79


title: 7E_M_86


title: 7E_M_87


title: 7E_M_83


title: 7E_M_85


title: 7E_M_77


title: 7E_M_78


title: 7E_M_88


title: 7E_M_75


title: 7E_M_76


title: 7E_M_80


title: 7E_M_74


title: 7E_M_72


title: 7E_M_73


title: 7E_M_71


title: 7E_M_68


title: 7E_M_66


title: 7E_M_70


title: 7E_M_67


title: 7E_M_69


title: 7E_M_82


title: 7E_M_63


title: 7E_M_65


title: 7E_M_2933


title: 7E_M_64


title: 7E_M_2932


title: 7E_M_2931


title: 7E_M_2930


title: 7E_M_2924


title: 7E_M_2929


title: 7E_M_2923


title: 7E_M_2927


title: 7E_M_2921


title: 7E_M_2922


title: 7E_M_2925


title: 7E_M_126


title: 7E_M_2917


title: 7E_M_2920


title: 7E_M_2918


title: 7E_M_2919


title: 7E_M_2915


title: 7E_M_2914


title: 7E_M_2913


title: 7E_M_2912


title: 7E_M_2909


title: 7E_M_2911


title: 7E_M_2910


title: 7E_M_2907


title: 7E_M_2906


title: 7E_M_2905


title: 7E_M_2903


title: 7E_M_2908


title: 7E_M_2904


title: 7E_M_2928


title: 7E_M_2926


title: 7E_M_2900


title: 7E_M_2901


title: 7E_M_2902


title: 7E_M_2896


title: 7E_M_2897


title: 7E_M_2898


title: 7E_M_2895


title: 7E_M_2916


title: 7E_M_2899


title: 7E_M_2894


title: 7E_M_2893


title: 7E_M_2892


title: 7E_M_2891


title: 7E_M_2889


title: 7E_M_2888


title: 7E_M_2885


title: 7E_M_2883


title: 7E_M_2887


title: 7E_M_2884


title: 7E_M_2886


title: 7E_M_2882


title: 7E_M_2879


title: 7E_M_2876


title: 7E_M_2878


title: 7E_M_2874


title: 7E_M_2881


title: 7E_M_2880


title: 7E_M_2875


title: 7E_M_2877


title: 7E_M_2873


title: 7E_M_2613


title: 7E_M_2871


title: 7E_M_2890


title: 7E_M_2609


title: 7E_M_2608


title: 7E_M_2870


title: 7E_M_2614


title: 7E_M_2872


title: 7E_M_2606


title: 7E_M_2605


title: 7E_M_2612


title: 7E_M_2607


title: 7E_M_2610


title: 7E_M_2611


title: 7E_M_2604


title: 7E_M_2602


title: 7E_M_2601


title: 7E_M_2603


title: 7E_M_2598


title: 7E_M_2600


title: 7E_M_2599


title: 7E_M_2596


title: 7E_M_2595


title: 7E_M_2593


title: 7E_M_2594


title: 7E_M_2590


title: 7E_M_2592


title: 7E_M_2589


title: 7E_M_2585


title: 7E_M_2591


title: 7E_M_2580


title: 7E_M_2581


title: 7E_M_2584


title: 7E_M_2587


title: 7E_M_2577


title: 7E_M_2582


title: 7E_M_2586


title: 7E_M_2578


title: 7E_M_2575


title: 7E_M_2588


title: 7E_M_2576


title: 7E_M_2574


title: 7E_M_2573


title: 7E_M_2570


title: 7E_M_2571


title: 7E_M_2597


title: 7E_M_2569


title: 7E_M_2566


title: 7E_M_2565


title: 7E_M_2563


title: 7E_M_2564


title: 7E_M_2560


title: 7E_M_2562


title: 7E_M_2567


title: 7E_M_2568


title: 7E_M_2561


title: 7E_M_2559


title: 7E_M_2579


title: 7E_M_2555


title: 7E_M_2557


title: 7E_M_2572


title: 7E_M_2554


title: 7E_M_2556


title: 7E_M_2551


title: 7E_M_2423


title: 7E_M_2553


title: 7E_M_2420


title: 7E_M_2419


title: 7E_M_2418


title: 7E_M_2558


title: 7E_M_2422


title: 7E_M_2421


title: 7E_M_2417


title: 7E_M_2416


title: 7E_M_2415


title: 7E_M_2413


title: 7E_M_2412


title: 7E_M_2410


title: 7E_M_2414


title: 7E_M_2406


title: 7E_M_2407


title: 7E_M_2552


title: 7E_M_2411


title: 7E_M_2408


title: 7E_M_2405


title: 7E_M_2409


title: 7E_M_2404


title: 7E_M_2403


title: 7E_M_2390


title: 7E_M_2391


title: 7E_M_2402


title: 7E_M_2396


title: 7E_M_2397


title: 7E_M_2398


title: 7E_M_2401


title: 7E_M_2395


title: 7E_M_2400


title: 7E_M_2386


title: 7E_M_2385


title: 7E_M_2393


title: 7E_M_2399


title: 7E_M_2394


title: 7E_M_2383


title: 7E_M_2384


title: 7E_M_2376


title: 7E_M_2382


title: 7E_M_2375


title: 7E_M_2374


title: 7E_M_2378


title: 7E_M_2389


title: 7E_M_2388


title: 7E_M_2377


title: 7E_M_2381


title: 7E_M_2371


title: 7E_M_2380


title: 7E_M_2366


title: 7E_M_2168


title: 7E_M_2362


title: 7E_M_2364


title: 7E_M_2370


title: 7E_M_2167


title: 7E_M_2373


title: 7E_M_2367


title: 7E_M_2163


title: 7E_M_2165


title: 7E_M_2166


title: 7E_M_2361


title: 7E_M_2365


title: 7E_M_2164


title: 7E_M_2379


title: 7E_M_2169


title: 7E_M_2162


title: 7E_M_2161


title: 7E_M_2158


title: 7E_M_2160


title: 7E_M_2157


title: 7E_M_2156


title: 7E_M_2363


title: 7E_M_2372


title: 7E_M_2368


title: 7E_M_2155


title: 7E_M_2369


title: 7E_M_2154


title: 7E_M_2150


title: 7E_M_2149


title: 7E_M_2148


title: 7E_M_2152


title: 7E_M_2146


title: 7E_M_2151


title: 7E_M_2153


title: 7E_M_2137


title: 7E_M_2144


title: 7E_M_2139


title: 7E_M_2141


title: 7E_M_2138


title: 7E_M_2159


title: 7E_M_2145


title: 7E_M_2133


title: 7E_M_2132


title: 7E_M_2131


title: 7E_M_2135


title: 7E_M_2134


title: 7E_M_2126


title: 7E_M_2130


title: 7E_M_2129


title: 7E_M_2128


title: 7E_M_2127


title: 7E_M_2147


title: 7E_M_2143


title: 7E_M_2142


title: 7E_M_2140


title: 7E_M_2136


title: 7E_M_2122


title: 7E_M_2125


title: 7E_M_2120


title: 7E_M_2119


title: 7E_M_2123


title: 7E_M_2118


title: 7E_M_2115


title: 7E_M_2116


title: 7E_M_2112


title: 7E_M_2124


title: 7E_M_2113


title: 7E_M_2117


title: 7E_M_2121


title: 7E_M_2106


title: 7E_M_2108


title: 7E_M_2107


title: 7E_M_2109


title: 7E_M_2105


title: 7E_M_2103


title: 7E_M_2104


title: 7E_M_2102


title: 7E_M_2114


title: 7E_M_2098


title: 7E_M_2101


title: 7E_M_2099


title: 7E_M_2097


title: 7E_M_2100


title: 7E_M_2096


title: 7E_M_2111


title: 7E_M_2110


title: 7E_M_2093


title: 7E_M_2095


title: 7E_M_2094


title: 7E_M_2090


title: 7E_M_2092


title: 7E_M_2089


title: 7E_M_2087


title: 7E_M_2088


title: 7E_M_2086


title: 7E_M_2085


title: 7E_M_2083


title: 7E_M_2081


title: 7E_M_2077


title: 7E_M_2079


title: 7E_M_2080


title: 7E_M_2078


title: 7E_M_2074


title: 7E_M_2076


title: 7E_M_2075


title: 7E_M_2068


title: 7E_M_2070


title: 7E_M_2072


title: 7E_M_2069


title: 7E_M_2067


title: 7E_M_2066


title: 7E_M_2091


title: 7E_M_2062


title: 7E_M_2065


title: 7E_M_2063


title: 7E_M_2064


title: 7E_M_2084


title: 7E_M_2060


title: 7E_M_2061


title: 7E_M_2059


title: 7E_M_2057


title: 7E_M_2054


title: 7E_M_2053


title: 7E_M_2073


title: 7E_M_2056


title: 7E_M_2051


title: 7E_M_2058


title: 7E_M_2052


title: 7E_M_2049


title: 7E_M_2055


title: 7E_M_2048


title: 7E_M_2045


title: 7E_M_2047


title: 7E_M_2046


title: 7E_M_2043


title: 7E_M_2042


title: 7E_M_1912


title: 7E_M_2044


title: 7E_M_1910


title: 7E_M_1911


title: 7E_M_1909


title: 7E_M_1907


title: 7E_M_1904


title: 7E_M_1905


title: 7E_M_1906


title: 7E_M_1903


title: 7E_M_1902


title: 7E_M_1900


title: 7E_M_2050


title: 7E_M_1897


title: 7E_M_1899


title: 7E_M_1898


title: 7E_M_1894


title: 7E_M_1895


title: 7E_M_1892


title: 7E_M_1891


title: 7E_M_1890


title: 7E_M_1908


title: 7E_M_1893


title: 7E_M_1901


title: 7E_M_1889


title: 7E_M_1887


title: 7E_M_1888


title: 7E_M_1886


title: 7E_M_1885


title: 7E_M_1884


title: 7E_M_1883


title: 7E_M_1880


title: 7E_M_1882


title: 7E_M_1881


title: 7E_M_1879


title: 7E_M_1878


title: 7E_M_1877


title: 7E_M_1876


title: 7E_M_1896


title: 7E_M_1875


title: 7E_M_1874


title: 7E_M_1873


title: 7E_M_1872


title: 7E_M_1870


title: 7E_M_1869


title: 7E_M_1871


title: 7E_M_1868


title: 7E_M_1865


title: 7E_M_1866


title: 7E_M_1867


title: 7E_M_1859


title: 7E_M_1864


title: 7E_M_1858


title: 7E_M_1862


title: 7E_M_1857


title: 7E_M_1860


title: 7E_M_1856


title: 7E_M_1855


title: 7E_M_1852


title: 7E_M_1851


title: 7E_M_1854


title: 7E_M_1853


title: 7E_M_1787


title: 7E_M_1786


title: 7E_M_1785


title: 7E_M_1783


title: 7E_M_1784


title: 7E_M_1782


title: 7E_M_1781


title: 7E_M_1863


title: 7E_M_1861


title: 7E_M_1778


title: 7E_M_1779


title: 7E_M_1773


title: 7E_M_1772


title: 7E_M_1776


title: 7E_M_1774


title: 7E_M_1775


title: 7E_M_1771


title: 7E_M_1770


title: 7E_M_1766


title: 7E_M_1767


title: 7E_M_1768


title: 7E_M_1769


title: 7E_M_1780


title: 7E_M_1764


title: 7E_M_1765


title: 7E_M_1761


title: 7E_M_1763


title: 7E_M_1762


title: 7E_M_1759


title: 7E_M_1760


title: 7E_M_1758


title: 7E_M_1755


title: 7E_M_1754


title: 7E_M_1753


title: 7E_M_1752


title: 7E_M_1757


title: 7E_M_1750


title: 7E_M_1751


title: 7E_M_1777


title: 7E_M_1749


title: 7E_M_1748


title: 7E_M_1746


title: 7E_M_1745


title: 7E_M_1747


title: 7E_M_1742


title: 7E_M_1744


title: 7E_M_1741


title: 7E_M_1736


title: 7E_M_1739


title: 7E_M_1735


title: 7E_M_1738


title: 7E_M_1740


title: 7E_M_1732


title: 7E_M_1737


title: 7E_M_1729


title: 7E_M_1733


title: 7E_M_1734


title: 7E_M_1730


title: 7E_M_1727


title: 7E_M_1728


title: 7E_M_1726


title: 7E_M_1731


title: 7E_M_1659


title: 7E_M_1725


title: 7E_M_1656


title: 7E_M_1657


title: 7E_M_1743


title: 7E_M_1655


title: 7E_M_1658


title: 7E_M_1653


title: 7E_M_1650


title: 7E_M_1654


title: 7E_M_1648


title: 7E_M_1644


title: 7E_M_1646


title: 7E_M_1724


title: 7E_M_1643


title: 7E_M_1647


title: 7E_M_1645


title: 7E_M_1641


title: 7E_M_1639


title: 7E_M_1638


title: 7E_M_1636


title: 7E_M_1652


title: 7E_M_1642


title: 7E_M_1634


title: 7E_M_1640


title: 7E_M_1635


title: 7E_M_1633


title: 7E_M_1637


title: 7E_M_1631


title: 7E_M_1632


title: 7E_M_1628


title: 7E_M_1629


title: 7E_M_1630


title: 7E_M_1651


title: 7E_M_1649


title: 7E_M_1627


title: 7E_M_1626


title: 7E_M_1623


title: 7E_M_1625


title: 7E_M_1624


title: 7E_M_1619


title: 7E_M_1617


title: 7E_M_1615


title: 7E_M_1618


title: 7E_M_1616


title: 7E_M_1614


title: 7E_M_1612


title: 7E_M_1613


title: 7E_M_1607


title: 7E_M_1610


title: 7E_M_1608


title: 7E_M_1606


title: 7E_M_1611


title: 7E_M_1605


title: 7E_M_1602


title: 7E_M_1603


title: 7E_M_1601


title: 7E_M_1599


title: 7E_M_1598


title: 7E_M_1600


title: 7E_M_1622


title: 7E_M_1596


title: 7E_M_1621


title: 7E_M_1620


title: 7E_M_1595


title: 7E_M_1609


title: 7E_M_1592


title: 7E_M_1591


title: 7E_M_1604


title: 7E_M_1586


title: 7E_M_1587


title: 7E_M_1585


title: 7E_M_1588


title: 7E_M_1584


title: 7E_M_1580


title: 7E_M_1583


title: 7E_M_1581


title: 7E_M_1597


title: 7E_M_1579


title: 7E_M_1582


title: 7E_M_1576


title: 7E_M_1573


title: 7E_M_1578


title: 7E_M_1572


title: 7E_M_1571


title: 7E_M_1574


title: 7E_M_1594


title: 7E_M_1575


title: 7E_M_1593


title: 7E_M_1577


title: 7E_M_1570


title: 7E_M_1569


title: 7E_M_1568


title: 7E_M_1566


title: 7E_M_1590


title: 7E_M_1567


title: 7E_M_1589


title: 7E_M_1564


title: 7E_M_1558


title: 7E_M_1559


title: 7E_M_1555


title: 7E_M_1553


title: 7E_M_1554


title: 7E_M_1552


title: 7E_M_1551


title: 7E_M_1563


title: 7E_M_1550


title: 7E_M_1549


title: 7E_M_1543


title: 7E_M_1544


title: 7E_M_1542


title: 7E_M_1545


title: 7E_M_1548


title: 7E_M_1546


title: 7E_M_1547


title: 7E_M_1560


title: 7E_M_1557


title: 7E_M_1540


title: 7E_M_1561


title: 7E_M_1541


title: 7E_M_1539


title: 7E_M_1537


title: 7E_M_1562


title: 7E_M_1538


title: 7E_M_1536


title: 7E_M_1565


title: 7E_M_1535


title: 7E_M_1556


title: 7E_M_1534


title: 7E_M_1529


title: 7E_M_1533


title: 7E_M_1531


title: 7E_M_1532


title: 7E_M_1528


title: 7E_M_1527


title: 7E_M_1526


title: 7E_M_1525


title: 7E_M_1522


title: 7E_M_1523


title: 7E_M_1521


title: 7E_M_1519


title: 7E_M_1520


title: 7E_M_1518


title: 7E_M_1517


title: 7E_M_1510


title: 7E_M_1511


title: 7E_M_1516


title: 7E_M_1512


title: 7E_M_1509


title: 7E_M_1514


title: 7E_M_1507


title: 7E_M_1515


title: 7E_M_1513


title: 7E_M_1505


title: 7E_M_1506


title: 7E_M_1508


title: 7E_M_1530


title: 7E_M_1524


title: 7E_M_1503


title: 7E_M_1504


title: 7E_M_1499


title: 7E_M_1500


title: 7E_M_1498


title: 7E_M_1497


title: 7E_M_1495


title: 7E_M_1496


title: 7E_M_1494


title: 7E_M_1493


title: 7E_M_1491


title: 7E_M_1490


title: 7E_M_1492


title: 7E_M_1488


title: 7E_M_1486


title: 7E_M_1489


title: 7E_M_1487


title: 7E_M_1480


title: 7E_M_1482


title: 7E_M_1485


title: 7E_M_1501


title: 7E_M_1481


title: 7E_M_1483


title: 7E_M_1484


title: 7E_M_1502


title: 7E_M_1479


title: 7E_M_1472


title: 7E_M_1475


title: 7E_M_1476


title: 7E_M_1477


title: 7E_M_1478


title: 7E_M_1474


title: 7E_M_1469


title: 7E_M_1466


title: 7E_M_1467


title: 7E_M_1468


title: 7E_M_1471


title: 7E_M_1465


title: 7E_M_1470


title: 7E_M_1463


title: 7E_M_1464


title: 7E_M_1462


title: 7E_M_1461


title: 7E_M_1460


title: 7E_M_1458


title: 7E_M_1457


title: 7E_M_1451


title: 7E_M_1456


title: 7E_M_1455


title: 7E_M_1452


title: 7E_M_1459


title: 7E_M_1454


title: 7E_M_1448


title: 7E_M_1473


title: 7E_M_1445


title: 7E_M_1450


title: 7E_M_1449


title: 7E_M_1444


title: 7E_M_1447


title: 7E_M_1446


title: 7E_M_1442


title: 7E_M_1443


title: 7E_M_1440


title: 7E_M_1439


title: 7E_M_1441


title: 7E_M_1437


title: 7E_M_1438


title: 7E_M_1435


title: 7E_M_1434


title: 7E_M_1433


title: 7E_M_1453


title: 7E_M_1432


title: 7E_M_1431


title: 7E_M_1430


title: 7E_M_1426


title: 7E_M_1428


title: 7E_M_1429


title: 7E_M_1423


title: 7E_M_1424


title: 7E_M_1421


title: 7E_M_1417


title: 7E_M_1419


title: 7E_M_1436


title: 7E_M_1422


title: 7E_M_1420


title: 7E_M_1411


title: 7E_M_1410


title: 7E_M_1408


title: 7E_M_1414


title: 7E_M_1416


title: 7E_M_1427


title: 7E_M_1425


title: 7E_M_1415


title: 7E_M_1412


title: 7E_M_1418


title: 7E_M_1407


title: 7E_M_1409


title: 7E_M_1405


title: 7E_M_1404


title: 7E_M_1274


title: 7E_M_1275


title: 7E_M_1273


title: 7E_M_1271


title: 7E_M_1272


title: 7E_M_1413


title: 7E_M_1270


title: 7E_M_1269


title: 7E_M_1267


title: 7E_M_1264


title: 7E_M_1268


title: 7E_M_1262


title: 7E_M_1265


title: 7E_M_1260


title: 7E_M_1266


title: 7E_M_1259


title: 7E_M_1254


title: 7E_M_1255


title: 7E_M_1253


title: 7E_M_1256


title: 7E_M_1406


title: 7E_M_1258


title: 7E_M_1261


title: 7E_M_1252


title: 7E_M_1257


title: 7E_M_1250


title: 7E_M_1247


title: 7E_M_1249


title: 7E_M_1244


title: 7E_M_1246


title: 7E_M_1245


title: 7E_M_1243


title: 7E_M_1263


title: 7E_M_1242


title: 7E_M_1241


title: 7E_M_1240


title: 7E_M_1238


title: 7E_M_1239


title: 7E_M_1237


title: 7E_M_1235


title: 7E_M_1236


title: 7E_M_1233


title: 7E_M_1234


title: 7E_M_1231


title: 7E_M_1230


title: 7E_M_1232


title: 7E_M_1229


title: 7E_M_1226


title: 7E_M_1228


title: 7E_M_1225


title: 7E_M_1227


title: 7E_M_1223


title: 7E_M_1221


title: 7E_M_1222


title: 7E_M_1219


title: 7E_M_1218


title: 7E_M_1220


title: 7E_M_1216


title: 7E_M_1217


title: 7E_M_1213


title: 7E_M_1214


title: 7E_M_1215


title: 7E_M_1212


title: 7E_M_1209


title: 7E_M_1210


title: 7E_M_1208


title: 7E_M_1207


title: 7E_M_1204


title: 7E_M_1205


title: 7E_M_1206


title: 7E_M_1224


title: 7E_M_1203


title: 7E_M_1202


title: 7E_M_1198


title: 7E_M_1201


title: 7E_M_1197


title: 7E_M_1196


title: 7E_M_1200


title: 7E_M_1199


title: 7E_M_1195


title: 7E_M_1192


title: 7E_M_1193


title: 7E_M_1194


title: 7E_M_1191


title: 7E_M_1188


title: 7E_M_1187


title: 7E_M_1211


title: 7E_M_1186


title: 7E_M_1184


title: 7E_M_1183


title: 7E_M_1185


title: 7E_M_1181


title: 7E_M_1182


title: 7E_M_1180


title: 7E_M_1179


title: 7E_M_1175


title: 7E_M_1177


title: 7E_M_1178


title: 7E_M_1170


title: 7E_M_1171


title: 7E_M_1172


title: 7E_M_1176


title: 7E_M_1169


title: 7E_M_1189


title: 7E_M_1167


title: 7E_M_1174


title: 7E_M_1168


title: 7E_M_1173


title: 7E_M_1190


title: 7E_M_1162


title: 7E_M_1164


title: 7E_M_1165


title: 7E_M_1161


title: 7E_M_1159


title: 7E_M_1160


title: 7E_M_1158


title: 7E_M_1156


title: 7E_M_1157


title: 7E_M_1154


title: 7E_M_1153


title: 7E_M_1150


title: 7E_M_1151


title: 7E_M_1166


title: 7E_M_1149


title: 7E_M_1148


title: 7E_M_1083


title: 7E_M_1081


title: 7E_M_1080


title: 7E_M_1079


title: 7E_M_1163


title: 7E_M_1077


title: 7E_M_1071


title: 7E_M_1078


title: 7E_M_1076


title: 7E_M_1073


title: 7E_M_1070


title: 7E_M_1069


title: 7E_M_1155


title: 7E_M_1067


title: 7E_M_1152


title: 7E_M_1068


title: 7E_M_1063


title: 7E_M_1066


title: 7E_M_1064


title: 7E_M_1062


title: 7E_M_1061


title: 7E_M_1082


title: 7E_M_1075


title: 7E_M_1054


title: 7E_M_1060


title: 7E_M_1057


title: 7E_M_1058


title: 7E_M_1056


title: 7E_M_1055


title: 7E_M_1059


title: 7E_M_1072


title: 7E_M_1053


title: 7E_M_1052


title: 7E_M_1051


title: 7E_M_1050


title: 7E_M_1074


title: 7E_M_1047


title: 7E_M_1049


title: 7E_M_1045


title: 7E_M_1044


title: 7E_M_1065


title: 7E_M_1046


title: 7E_M_1042


title: 7E_M_1040


title: 7E_M_1041


title: 7E_M_1037


title: 7E_M_1039


title: 7E_M_1036


title: 7E_M_1034


title: 7E_M_1035


title: 7E_M_1048


title: 7E_M_1026


title: 7E_M_1031


title: 7E_M_1030


title: 7E_M_1032


title: 7E_M_1027


title: 7E_M_1043


title: 7E_M_1024


title: 7E_M_1025


title: 7E_M_1023


title: 7E_M_956


title: 7E_M_1022


title: 7E_M_1021


title: 7E_M_954


title: 7E_M_955


title: 7E_M_951


title: 7E_M_953


title: 7E_M_1020


title: 7E_M_950


title: 7E_M_949


title: 7E_M_947


title: 7E_M_1038


title: 7E_M_948


title: 7E_M_944


title: 7E_M_1033


title: 7E_M_945


title: 7E_M_946


title: 7E_M_1029


title: 7E_M_1028


title: 7E_M_938


title: 7E_M_941


title: 7E_M_952


title: 7E_M_940


title: 7E_M_937


title: 7E_M_939


title: 7E_M_931


title: 7E_M_935


title: 7E_M_936


title: 7E_M_942


title: 7E_M_943


title: 7E_M_933


title: 7E_M_929


title: 7E_M_927


title: 7E_M_928


title: 7E_M_926


title: 7E_M_924


title: 7E_M_930


title: 7E_M_923


title: 7E_M_922


title: 7E_M_919


title: 7E_M_918


title: 7E_M_921


title: 7E_M_917


title: 7E_M_915


title: 7E_M_916


title: 7E_M_934


title: 7E_M_914


title: 7E_M_911


title: 7E_M_932


title: 7E_M_913


title: 7E_M_910


title: 7E_M_909


title: 7E_M_908


title: 7E_M_907


title: 7E_M_905


title: 7E_M_906


title: 7E_M_904


title: 7E_M_903


title: 7E_M_899


title: 7E_M_901


title: 7E_M_900


title: 7E_M_896


title: 7E_M_920


title: 7E_M_902


title: 7E_M_897


title: 7E_M_895


title: 7E_M_894


title: 7E_M_892


title: 7E_M_891


title: 7E_M_890


title: 7E_M_912


title: 7E_M_893


title: 7E_M_898


title: 7E_M_889


title: 7E_M_888


title: 7E_M_885


title: 7E_M_886


title: 7E_M_883


title: 7E_M_884


title: 7E_M_887


title: 7E_M_882


title: 7E_M_881


title: 7E_M_879


title: 7E_M_878


title: 7E_M_875


title: 7E_M_876


title: 7E_M_877


title: 7E_M_872


title: 7E_M_868


title: 7E_M_874


title: 7E_M_870


title: 7E_M_871


title: 7E_M_866


title: 7E_M_867


title: 7E_M_865


title: 7E_M_864


title: 7E_M_863


title: 7E_M_862


title: 7E_M_861


title: 7E_M_860


title: 7E_M_859


title: 7E_M_856


title: 7E_M_880


title: 7E_M_857


title: 7E_M_854


title: 7E_M_858


title: 7E_M_855


title: 7E_M_869


title: 7E_M_853


title: 7E_M_851


title: 7E_M_850


title: 7E_M_873


title: 7E_M_849


title: 7E_M_848


title: 7E_M_847


title: 7E_M_846


title: 7E_M_845


title: 7E_M_842


title: 7E_M_843


title: 7E_M_841


title: 7E_M_837


title: 7E_M_838


title: 7E_M_840


title: 7E_M_835


title: 7E_M_839


title: 7E_M_833


title: 7E_M_832


title: 7E_M_834


title: 7E_M_836


title: 7E_M_852


title: 7E_M_828


title: 7E_M_827


title: 7E_M_826


title: 7E_M_824


title: 7E_M_830


title: 7E_M_825


title: 7E_M_831


title: 7E_M_829


title: 7E_M_822


title: 7E_M_821


title: 7E_M_823


title: 7E_M_820


title: 7E_M_819


title: 7E_M_844


title: 7E_M_817


title: 7E_M_816


title: 7E_M_815


title: 7E_M_813


title: 7E_M_812


title: 7E_M_814


title: 7E_M_811


title: 7E_M_818


title: 7E_M_808


title: 7E_M_810


title: 7E_M_807


title: 7E_M_809


title: 7E_M_806


title: 7E_M_805


title: 7E_M_804


title: 7E_M_803


title: 7E_M_802


title: 7E_M_801


title: 7E_M_800


title: 7E_M_798


title: 7E_M_797


title: 7E_M_796


title: 7E_M_799


title: 7E_M_795


title: CdGAP3: CdGAP fx/fx tumor rep 3

city: Boston

first: Marie-Anne

last: Goyette

address: 450 Brookline avenue

institute: DFCI

library_source: transcriptomic

data_processing:
Quality of the raw reads was assessed with FastQC 0.11.5
Reads were aligned to the GRCm38 genome with Star 2.5.1b
Raw alignment counts were calculated with featureCounts 1.4.6
Differential expression measurements were performed with DESeq2 1.12.3
Illumina HiSeq 2000
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text file with raw alignment counts of each sample
Supplementary_files_format_and_content: excel file of Deseq analysis including raw counts normalized relative to the total number of reads

molecule: total RNA

source_name: NIC+ tumors

extract_protocol:
Total RNA was extracted using TRIZOL reagent (Invitrogen) and purified on RNeasy columns (Qiagen)
RNA libraries were prepared for sequencing using standard Illumina protocols

value: NIC+ tumors

value: CdGAP fx/fx


title: 7E_M_794


title: CdGAP2: CdGAP fx/fx tumor rep 2


title: Nb_rep 1

city: Tübingen

first: Chenlei

last: Hua

address: Auf der Morgenstelle 32

department: Department of Plant Biochemistry, Centre of Plant Molecular Biology (ZMBP)

country: Germany

institute: University of Tübingen

data_processing:
Primary sequencing data that produced by Illumina HiSeqTM 2000, called as raw reads, is subjected to quality control (QC) that determine if a resequencing step is needed.
After QC, raw reads are filtered into clean reads which will be aligned to the reference sequences.
QC of alignment is performed to determine if resequencing is needed. The alignment data is utilized to calculate distribution of reads on reference genes and perform coverage analysis.
Results of gene expression include gene expression levels and differential expression analysis.
Further, we perform Gene Ontology (GO) enrichment analysis and Pathway enrichment analysis.
Assembly: GCA_000723945.1Ni_benscaffolds:100,480contigs:100,480N50:776L50:22,990
Supplementary files format and content: .xls file containing all the gene expression reads and relative expression levels

organism: Nicotiana benthamiana

source_name: seedlings

extract_protocol:
Total RNA was isolated using a Nucleospin® RNA Plant Kit (Macherey-Nagel, Germany) according to the manufacturer’s instructions.
After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA or by removing rRNAs from the total RNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates.

value: seedlings


title: Nb_rep 2


title: 7E_M_793


title: 7E_M_791


title: WT3: CdGAP wt/wt tumor rep 3

value: CdGAP wt/wt


title: 7E_M_792


title: WT1: CdGAP wt/wt tumor rep 1


title: 7E_M_790


title: 7E_M_789


title: org_adult_rep3_bisulfite (small intestinal epithelium derived organoids)

city: Copenhagen N

first: Laura

middle: Maarit

last: Pikkupeura

address: Ole Maaloes vej 5

department: BRIC

country: Denmark

institute: University of Copenhagen

data_processing:
Reads were quality trimmed with Trim Galore.
Reads were mapped and methylation status was determined with Bismark, which bisulfite converts the reads in silico prior to mapping with Bowtie2 to a bisulfite converted reference genome. (Krueger and Andrews, 2011). The mapping efficiency was low due to large number of chimeric reads. To mitigate this, the mapping was performed in two steps: first all reads were mapped in paired end mode with the option --unmapped, following which the unmapped R1 were mapped in single-end mode (directional mode) and the unmapped R2 in single-end mode (--pbat mode). The paired-end and single-end alignments were merged together after methylation extraction.
To segregate the genome into PMDs and non-PMDs, we use MethylSeekR (Burger et al., 2013). We identified a consensus set of PMDs present in all 6 samples. Fetal and adult state specific PMDs were defined as regions present in all replicates in one condition and none of the replicates in the other condition. We identified differentially methylated regions by BSmooth implemented in bsseq (Hansen et al., 2012). We used the methylation calls processed with Bismark and performed the smoothing and differential methylation analysis with BSmooth. We used only CpGs which were covered by more than four reads in all six samples. The smoothing was done for regions containing at least 70 CpG or were at least 2kb wide, whichever is largest, using the whole chromosomes as the smoothing cluster. For computing t-statistics, the variability was estimated based on the fetal samples. For identifying differentially methylated regions we used a cutoff 4.6, based on t-statistic quantiles.
Integrative Genomics Viewer (IGV) viewable TGF where the score represents percentage of methylated cytosines per-base CpG.
Genome_build: mm10

growth_protocol: Fetal spheroids and adult organoids derived from E16.5 fetal epithelial fragments or scraped adult crypts harvested from proximal small intestine using 2mM EDTA were embedded in Matrigel droplets and cultured in advanced DMEM/F12 with GlutaMAX and Penicillin/Streptomycin supplemented with 50 ng/ml EGF, 100 ng/ml Noggin, and 500 ng/ml, R-Spondin1, 3 µM CHIR99021 and 10 mM Nicotinamide.

extract_protocol:
Genomic DNA of fetal (3 independent biological replicates) and adult organoids (3 independent biological replicates) was purified using Genomic DNA Purification Kit (Thermo scientific)
Libraries were prepared with ACCEL-NGS METHYL-SEQ DNA Library Kit.

tag: developmental state

value: Adult

tag: treatmet

value: untreated


title: CdGAP1: CdGAP fx/fx tumor rep 1


title: 7E_M_788


title: 7E_M_787


title: org_adult_rep2_bisulfite (small intestinal epithelium derived organoids)


title: org_fetal_rep3_bisulfite (small intestinal epithelium derived organoids)


title: org_adult_rep1_bisulfite (small intestinal epithelium derived organoids)


title: 7E_M_785


title: org_fetal_rep2_bisulfite (small intestinal epithelium derived organoids)


title: 7E_M_786


title: 7E_M_784


title: 7E_M_782


title: org_adult_rep3_cage

data_processing:
Sequencing reads were converted by using bcf2fastq and demultiplexed according to the indexes
After removal of linker sequences and filtering, the reads were mapped to the mm10 assembly with Bowtie2 and only uniquely mapping reads not mapping to chrM were retained for further analysis.
5’ ends of CAGE tags (CTSSs) supported by only a single read were excluded from the analysis. CTSSs were subsequently Tags Per Million (TPM) normalised (CAGE reads per total mapped reads in library times 106). CTSSs on the same strand within 20bp of each other were merged into tag clusters (TCs) and quantified in all samples (single read CTSSs were included for the quantification). A summit was identified in each TC, defined as the single base-pair position within the TC with highest total TPM coverage across all samples.
Genome_build: mm10
Supplementary_files_format_and_content:  CAGE bigwig files where the score represents strand-specific per-base genome coverage normalized to the library size.

source_name: small intestinal epithelium derived organoids

extract_protocol:
Total RNA was isolated from established fetal and adult organoid cultures using Qiagen® RNEasy MicroKit.
CAGE libraries for three biological replicates of fetal spheroids and adult organoids were prepared as previously described (Takahashi et al., 2012, Nat Protoc, doi:10.1038/nprot.2012.005) with an input of 3000 ng of total RNA. After individually preparing CAGE libraries for each sample, three samples with unique barcodes (CTT, GAT, and ACG) were pooled per sequencing lane and 30% Phi-X was spiked-in to each lane. Sequencing was done with Illumina HiSeq2000.


title: org_fetal_rep1_bisulfite (small intestinal epithelium derived organoids)


title: org_adult_rep1_cage

value: small intestinal epithelium derived organoids


title: UCSF schwannoma #33

type: genomic

city: Stanford

first: David

middle: A.

last: Solomon

address: 300 Pasteur Dr, Lane L216

department: Pathology

institute: Stanford University School of Medicine

description: Schwannoma nerve sheath tumor

hyb_protocol: bisulfite converted DNA was amplified, fragmented, and hybridized to Illumina Infinium EPIC 850k Human DNA Methylation Beadchips using standard Illumina protocol

scan_protocol: Arrays were imaged using the Illumina iScan instrument following standard recommended Illumina scanner setting

data_processing: Methylation data were preprocessed using the minfi package (v.1.30.0) in R. The detection p-value for each sample was computed. Functional normalization with NOOB background correction and dye-bias normalization was performed.

label: Cy5 and Cy3

organism: Homo sapiens

source_name: Schwannoma

label_protocol: standard Illumina protocol

growth_protocol: Primary human tumor tissue was used as the source of genomic DNA for all patients.  No ex vivo culture or xenografting was performed.

extract_protocol: Tumor tissue was selectively punched from formalin-fixed, paraffin-embedded tissue blocks using using 2.0 mm disposable biopsy punches (Integra Miltex Instruments, cat# 33-31-P/25) or selectively scraped from unstained sections cut from formalin-fixed, paraffin-embedded tissue blocks.  Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer’s protocol.

treatment_protocol: Primary human tumor tissue was used as the source of genomic DNA for all patients at time of initial diagnosis before any radiation or chemotherapy had been administered.  No ex vivo culture or xenografting was performed.

tag: patient sex

tag: tumor location

value: Paraspinal nerve root

tag: oncogenic driver


title: UCSF schwannoma #17

description: Schwannoma nerve sheath tumor

value: Female

value: Vestibular nerve


title: UCSF schwannoma #15

description: Schwannoma nerve sheath tumor


title: 7E_M_783


title: org_adult_rep2_cage


title: UCSF schwannoma #34

description: Schwannoma nerve sheath tumor


title: 7E_M_780


title: 7E_M_779


title: org_fetal_rep3_cage


title: WT2: CdGAP wt/wt tumor rep 2


title: org_fetal_rep2_cage


title: 7E_M_778


title: 7E_M_777


title: org_fetal_rep1_cage


title: UCSF schwannoma #16

description: Schwannoma nerve sheath tumor


title: FM schwannoma #9

description: Schwannoma nerve sheath tumor

value: Median nerve


title: 7E_M_781


title: UCSF schwannoma #9

description: Schwannoma nerve sheath tumor


title: UCSF schwannoma #14

description: Schwannoma nerve sheath tumor

value: Sural nerve


title: 7E_M_773


title: 7E_M_776


title: Nb_Vd592_12d_rep 1

organism:
Nicotiana benthamiana
Verticillium dahliae


title: 7E_M_772


title: 7E_M_774


title: Nb_Vd592_12d_rep 2


title: UCSF schwannoma #2

description: Schwannoma nerve sheath tumor


title: UCSF schwannoma #31

description: Schwannoma nerve sheath tumor


title: 7E_M_775


title: Nb_Vd592_9d_rep 2


title: 7E_M_769


title: 7E_M_768


title: Nb_Vd592_6d_rep 2


title: UCSF schwannoma #26b

description: Schwannoma nerve sheath tumor


title: Nb_Vd592_6d_rep 1


title: 7E_M_770


title: Nb_Vd592_3d_rep 2


title: FM schwannoma #4

description: Schwannoma nerve sheath tumor


title: UCSF schwannoma #3

description: Schwannoma nerve sheath tumor

value: Spinal accessory nerve


title: Nb_Vd592_3d_rep 1


title: 7E_M_766


title: 7E_M_765


title: Nb_Vd171_12d_rep 2


title: 7E_M_771


title: UCSF schwannoma #4

description: Schwannoma nerve sheath tumor

value: Trigeminal nerve


title: Nb_Vd171_12d_rep 1


title: UCSF schwannoma #30

description: Schwannoma nerve sheath tumor


title: Nb_Vd592_9d_rep 1


title: Nb_Vd171_9d_rep 2


title: 7E_M_763


title: 7E_M_764


title: 7E_M_762


title: Nb_Vd171_9d_rep 1


title: 7E_M_761


title: Nb_Vd171_6d_rep 2


title: 7E_M_760


title: UCSF schwannoma #10

description: Schwannoma nerve sheath tumor

value: Superficial peroneal nerve


title: 7E_M_759


title: Nb_Vd171_3d_rep 1


title: 7E_M_767


title: Nb_Vd171_3d_rep 2


title: UCSF schwannoma #13

description: Schwannoma nerve sheath tumor

value: Male


title: UCSF schwannoma #11

description: Schwannoma nerve sheath tumor


title: FM schwannoma #3

description: Schwannoma nerve sheath tumor


title: 7E_M_756


title: FM schwannoma #1

description: Schwannoma nerve sheath tumor

value: Brachial plexus


title: 7E_M_757


title: Nb_Vd171_6d_rep 1

value: wild-type


title: 7E_M_755


title: WT_D_2B

city: Shanghai

first: Wen

last: Xiao

address: 320 Yueyang Road

department: Institute of Biochemistry and Cell Biology

country: China

institute: Shanghai Institutes for Biological Sciences, CAS

description:
WT_D_2B
P1KOtestis_processed_file.xlsx

data_processing:
The preprocessing steps of RNA-seq are as follows: The raw paired-end fastq reads were removed adapter and quality-filtered by TrimGalore-0.5.0 with the parameters --paired --quality 20 --phred33 --stringency 1 --length 35. The remaining reads were mapped to the genome or identified TE by STAR v2.9, respectively. Uniquely mapped reads and multiple mapped reads with fewer than 10,000 genome copies were used for downstream analysis. The specific alignment parameters are as follows: --winAnchorMultimapNmax 10000 --outFilterMultimapNmax 10000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --twopassMode Basic --outSAMprimaryFlag AllBestScore. Calculate the reads count of known genes or TEs by featureCounts v1.6.4 with the parameters -p -B -C -M -Q 20, The expression of known genes or TEs was normalized to the ERCC or mapped reads. Genes or TEs with normalized count>2 in 40% or more of samples were used for differential analysis. Differentially expressed genes or TEs were identified by applying combined thresholds on the basis of fold change (>4 or <0.25) and permutation test (Padj ≤ 0.01).
Assembly: hamster: BCM_Maur_2.0
Supplementary files format and content: P1KOtestis_processed_file.xlsx:tab-delimited text files include normalized values by ERCC and gene length for each Sample

organism: Mesocricetus auratus

source_name: testes

extract_protocol:
Samples were incubated at 72°C for 3 min to release and unfold the  RNA.
mRNA library construction:In brief, Samples were incubated at 72°C for 3 min to release the RNAs. For every single oocyte or embryo, 0.125 μL 1/100,000 dilution of the ERCC RNA Spike-In Mix (Invitrogen, Cat# 4456740) was used. Following reverse transcription and PCR pre-amplification, cDNA libraries were purified by Agencourt Ampure XP beads (0.8:1 ratio) (Beckman Coulter, Cat#A63881) and 2 ng of purified cDNA was used for a tagmentation reaction with Tn5 transposase. Amplified libraries were purified by Agencourt Ampure XP beads (1:1 ratio).

value: testes

value: male

tag: Stage

value: adult D

value: wild type

value: FACS


title: WT_D_2A

description:
WT_D_2A
P1KOtestis_processed_file.xlsx


title: UCSF schwannoma #12

description: Schwannoma nerve sheath tumor


title: UCSF schwannoma #1

description: Schwannoma nerve sheath tumor

value: Facial nerve


title: WT_D_1B

description:
WT_D_1B
P1KOtestis_processed_file.xlsx


title: 7E_M_754


title: 7E_M_758


title: WT_D_1A

description:
WT_D_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_753


title: WT_P_2A

description:
WT_P_2A
P1KOtestis_processed_file.xlsx

value: adult P


title: 7E_M_752


title: WT_P_2B

description:
WT_P_2B
P1KOtestis_processed_file.xlsx


title: 7E_M_751


title: WT_P_1B

description:
WT_P_1B
P1KOtestis_processed_file.xlsx


title: WT_P_1A

description:
WT_P_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_750


title: 7E_M_749


title: WT_LZ_2A

description:
WT_LZ_2A
P1KOtestis_processed_file.xlsx

value: adult LZ


title: 7E_M_747


title: WT_LZ_2B

description:
WT_LZ_2B
P1KOtestis_processed_file.xlsx


title: WT_LZ_1A

description:
WT_LZ_1A
P1KOtestis_processed_file.xlsx


title: WT_LZ_1B

description:
WT_LZ_1B
P1KOtestis_processed_file.xlsx


title: WT_SPG/PL_2B

description:
WT_SPG/PL_2B
P1KOtestis_processed_file.xlsx

value: adult SPG/PL


title: 7E_M_744


title: 7E_M_748


title: 7E_M_746


title: 7E_M_745


title: 7E_M_741


title: P1KO_D_2B

description:
P1KO_D_2B
P1KOtestis_processed_file.xlsx


title: WT_SPG/PL_2A

description:
WT_SPG/PL_2A
P1KOtestis_processed_file.xlsx


title: 7E_M_742


title: WT_SPG/PL_1B

description:
WT_SPG/PL_1B
P1KOtestis_processed_file.xlsx


title: WT_SPG/PL_1A

description:
WT_SPG/PL_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_739


title: 7E_M_740


title: 7E_M_737


title: P1KO_D_1A

description:
P1KO_D_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_736


title: P1KO_P_2B

description:
P1KO_P_2B
P1KOtestis_processed_file.xlsx


title: P1KO_P_2A

description:
P1KO_P_2A
P1KOtestis_processed_file.xlsx


title: 7E_M_735


title: P1KO_P_1B

description:
P1KO_P_1B
P1KOtestis_processed_file.xlsx


title: P1KO_P_1A

description:
P1KO_P_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_743


title: P1KO_D_2A

description:
P1KO_D_2A
P1KOtestis_processed_file.xlsx


title: 7E_M_732


title: P1KO_LZ_2A

description:
P1KO_LZ_2A
P1KOtestis_processed_file.xlsx


title: P1KO_LZ_2B

description:
P1KO_LZ_2B
P1KOtestis_processed_file.xlsx


title: 7E_M_733


title: 7E_M_731


title: P1KO_LZ_1B

description:
P1KO_LZ_1B
P1KOtestis_processed_file.xlsx


title: P1KO_SPG/PL_2B

description:
P1KO_SPG/PL_2B
P1KOtestis_processed_file.xlsx


title: P1KO_SPG/PL_1B

description:
P1KO_SPG/PL_1B
P1KOtestis_processed_file.xlsx


title: P1KO_D_1B

description:
P1KO_D_1B
P1KOtestis_processed_file.xlsx


title: 7E_M_729


title: 7E_M_730


title: 7E_M_726


title: 7E_M_728


title: F2R59 WT-ad-IgG-1

description:
WT-ad-IgG-1
smRNA_piRNA_file.zip

data_processing:
The preprocessing steps of smRNA-seq are as follows: intercept the first 50bp of reads and filter out low-quality sequences (-q 20, -p 90); After quality-filtering, sequencing reads were clipped from the 3′ adapter allowing for a minimum match of 10 nt from the 5′ end. Reads unable to match the adapter sequence or with lengths shorter than 17 bp were discarded, and the remaining reads were mapped to genome by bowtie (hamster: BCM_Maur_2.0, mouse: mm10), bowtie version 1.2.1.1, parameters -k=100 -v=0). If the reads mapped more than 100 loci to the genome, 100 of all mapped loci are randomly output. The reads were aligned sequentially to known miRNA, tRNA, rRNA, snoRNA, and snRNA sequences by Bowtie without any mismatch allowed (bowtie parameter is -k=100 -v=0 -norc), and reads that aligned to pre-miRNA were identified as miRNAs. The remaining reads of the specified length were extracted and used to identify piRNAs according to the previously described method (Jung et al. 2014) with slight modifications. The length of candidate piRNAs in male samples is 25~32nt, and the length of candidate piRNAs in female samples is 17~32nt. The clustering parameters were determined as MinReads = 5 and Eps = 2500 bp by running a series of k-dist analyses of our data with different Eps and MinReads. All of the candidate clusters that satisfied these parameters were considered piRNA clusters, and the remaining sequences located in these clusters were defined as piRNAs without any further filtering. Reads in each library were normalized by sequencing depth or reads count of miRNA or reads count of exogenous spike-in.
The preprocessing steps of RNA-seq are as follows: The raw paired-end fastq reads were removed adapter and quality-filtered by TrimGalore-0.5.0 with the parameters --paired --quality 20 --phred33 --stringency 1 --length 35. The remaining reads were mapped to the genome or identified TE by STAR v2.9, respectively. Uniquely mapped reads and multiple mapped reads with fewer than 10,000 genome copies were used for downstream analysis. The specific alignment parameters are as follows: --winAnchorMultimapNmax 10000 --outFilterMultimapNmax 10000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --twopassMode Basic --outSAMprimaryFlag AllBestScore. Calculate the reads count of known genes or TEs by featureCounts v1.6.4 with the parameters -p -B -C -M -Q 20, The expression of known genes or TEs was normalized to the ERCC or mapped reads. Genes or TEs with normalized count>2 in 40% or more of samples were used for differential analysis. Differentially expressed genes or TEs were identified by applying combined thresholds on the basis of fold change (≥2 or ≤0.5) and Welch two-sample t-test (P ≤ 0.05).
Assembly: hamster: BCM_Maur_2.0, mouse: mm10
Supplementary files format and content: testis_processed_file.xlsx and oocyte_processed_file.xlsx:tab-delimited text files include normalized values by ERCC and gene length for each Sample;
Supplementary files format and content: spermatogenesis_processed_file.xlsx:tab-delimited text files include normalized values by total reads and gene length for each Sample

extract_protocol:
Samples were incubated at 72°C for 3 min to release and unfold the small RNAs or RNA.
small RNA libraries construction: Briefly, Samples were incubated at 72°C for 3 min to release and unfold the small RNAs. After 3’ adapter ligation, 5U of lambda exonuclease and 25U of 5’ deadenylases, which performed better than the combination of RecJf and 5’ deadenylases, were used to remove the excess 3’ adaptor. Then, a 5’ adapter was ligated and small RNAs were reverse transcribed. Pre-amplification was performed and then 1 μl of amplified product was used as a template for final amplification. The amplified libraries were separated on a 6% polyacrylamide gel and 130-160 bp DNA fragments were sliced to recover the library of small RNAs.  mRNA library construction:In brief, Samples were incubated at 72°C for 3 min to release the RNAs. For every single oocyte or embryo, 0.125 μL 1/100,000 dilution of the ERCC RNA Spike-In Mix (Invitrogen, Cat# 4456740) was used. Following reverse transcription and PCR pre-amplification, cDNA libraries were purified by Agencourt Ampure XP beads (0.8:1 ratio) (Beckman Coulter, Cat#A63881) and 2 ng of purified cDNA was used for a tagmentation reaction with Tn5 transposase. Amplified libraries were purified by Agencourt Ampure XP beads (1:1 ratio).

value: adult

value: IgG IP


title: P1KO_SPG/PL_1A

description:
P1KO_SPG/PL_1A
P1KOtestis_processed_file.xlsx


title: 7E_M_727


title: 7E_M_724


title: 7E_M_734


title: 7E_M_723


title: F2R57 P4KO-ad-P2IP-1

description:
P4KO-ad-P2IP-1
smRNA_piRNA_file.zip

value: Piwil2 IP


title: F2R56 P3KO-ad-P2IP-2

description:
P3KO-ad-P2IP-2
smRNA_piRNA_file.zip


title: F2R55 P3KO-ad-P2IP-1

description:
P3KO-ad-P2IP-1
smRNA_piRNA_file.zip


title: F2R54 P2KO-ad-P2IP-2

description:
P2KO-ad-P2IP-2
smRNA_piRNA_file.zip

value: piwil2 -/-


title: 7E_M_719


title: 7E_M_720


title: F2R51 P1KO-ad-P2IP-1

description:
P1KO-ad-P2IP-1
smRNA_piRNA_file.zip


title: 7E_M_718


title: F2R52 P1KO-ad-P2IP-2

description:
P1KO-ad-P2IP-2
smRNA_piRNA_file.zip


title: 7E_M_717


title: F2R50 WT-ad-P2IP-2

description:
WT-ad-P2IP-2
smRNA_piRNA_file.zip


title: 7E_M_716


title: F2R42 P4KO-ad-P1IP-1

description:
P4KO-ad-P1IP-1
smRNA_piRNA_file.zip

value: Piwil1 IP


title: F2R45 WT-ad-P2IP-1

description:
WT-ad-P2IP-1
smRNA_piRNA_file.zip


title: F2R60 WT-ad-IgG-2

description:
WT-ad-IgG-2
smRNA_piRNA_file.zip


title: F2R41 P3KO-ad-P1IP-2

description:
P3KO-ad-P1IP-2
smRNA_piRNA_file.zip


title: 7E_M_712


title: F2R58 P4KO-ad-P2IP-2

description:
P4KO-ad-P2IP-2
smRNA_piRNA_file.zip


title: 7E_M_713


title: 7E_M_714


title: F2R43 P4KO-ad-P1IP-2

description:
P4KO-ad-P1IP-2
smRNA_piRNA_file.zip


title: 7E_M_725


title: F2R40 P3KO-ad-P1IP-1

description:
P3KO-ad-P1IP-1
smRNA_piRNA_file.zip


title: 7E_M_711


title: 7E_M_721


title: F2R39 P2KO-ad-P1IP-2

description:
P2KO-ad-P1IP-2
smRNA_piRNA_file.zip


title: 7E_M_710


title: 7E_M_722


title: 7E_M_709


title: F2R53 P2KO-ad-P2IP-1

description:
P2KO-ad-P2IP-1
smRNA_piRNA_file.zip


title: F2R38 P2KO-ad-P1IP-1

description:
P2KO-ad-P1IP-1
smRNA_piRNA_file.zip


title: 7E_M_708


title: F2R37 P1KO-ad-P1IP-2

description:
P1KO-ad-P1IP-2
smRNA_piRNA_file.zip


title: F2R36 P1KO-ad-P1IP-1

description:
P1KO-ad-P1IP-1
smRNA_piRNA_file.zip


title: 7E_M_715


title: 7E_M_706


title: 7E_M_705


title: F2R33 WT-3d-IgG-2

description:
WT-3d-IgG-2
smRNA_piRNA_file.zip


title: 7E_M_704


title: F2R32 WT-3d-IgG-1

description:
WT-3d-IgG-1
smRNA_piRNA_file.zip


title: F2R29 P4KO-3d-P4IP-1

description:
P4KO-3d-P4IP-1
smRNA_piRNA_file.zip

value: Piwil4 IP


title: F2R28 P2KO-3d-P4IP-2

description:
P2KO-3d-P4IP-2
smRNA_piRNA_file.zip


title: 7E_M_700


title: 7E_M_699


title: F2R27 P2KO-3d-P4IP-1

description:
P2KO-3d-P4IP-1
smRNA_piRNA_file.zip


title: 7E_M_698


title: F2R31 P4KO-3d-P4IP-2

description:
P4KO-3d-P4IP-2
smRNA_piRNA_file.zip


title: 7E_M_697


title: F2R24 P4KO-3d-P2IP-2

description:
P4KO-3d-P2IP-2
smRNA_piRNA_file.zip


title: 7E_M_696


title: F2R23 P4KO-3d-P2IP-1

description:
P4KO-3d-P2IP-1
smRNA_piRNA_file.zip


title: 7E_M_695


title: F2R25 WT-3d-P4IP-1

description:
WT-3d-P4IP-1
smRNA_piRNA_file.zip


title: 7E_M_707


title: 7E_M_694


title: F2R35 WT-ad-P1IP-2

description:
WT-ad-P1IP-2
smRNA_piRNA_file.zip


title: F2R34 WT-ad-P1IP-1

description:
WT-ad-P1IP-1
smRNA_piRNA_file.zip


title: 7E_M_693


title: 7E_M_702


title: F2R22 P2KO-3d-P2IP-2

description:
P2KO-3d-P2IP-2
smRNA_piRNA_file.zip


title: F2R21 P2KO-3d-P2IP-1

description:
P2KO-3d-P2IP-1
smRNA_piRNA_file.zip


title: 7E_M_692


title: F2R20 WT-3d-P2IP-2

description:
WT-3d-P2IP-2
smRNA_piRNA_file.zip


title: 7E_M_703


title: 7E_M_701


title: F2R19 WT-3d-P2IP-1

description:
WT-3d-P2IP-1
smRNA_piRNA_file.zip


title: 7E_M_690


title: N11N38 mature_sperm-2

description:
mature_sperm-2
spermatogenesis_processed_file.xlsx

data_processing:
The preprocessing steps of RNA-seq are as follows: The raw paired-end fastq reads were removed adapter and quality-filtered by TrimGalore-0.5.0 with the parameters --paired --quality 20 --phred33 --stringency 1 --length 35. The remaining reads were mapped to the genome or identified TE by STAR v2.9, respectively. Uniquely mapped reads and multiple mapped reads with fewer than 10,000 genome copies were used for downstream analysis. The specific alignment parameters are as follows: --winAnchorMultimapNmax 10000 --outFilterMultimapNmax 10000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --twopassMode Basic --outSAMprimaryFlag AllBestScore. Calculate the reads count of known genes or TEs by featureCounts v1.6.4 with the parameters -p -B -C -M -Q 20, The expression of known genes or TEs was normalized to the ERCC or mapped reads. Genes or TEs with normalized count>2 in 40% or more of samples were used for differential analysis. Differentially expressed genes or TEs were identified by applying combined thresholds on the basis of fold change (>4 or <0.25) and permutation test (Padj <= 0.01).
Assembly: hamster: BCM_Maur_2.0, mouse: mm10
Supplementary files format and content: testis_processed_file.xlsx and oocyte_processed_file.xlsx:tab-delimited text files include normalized values by ERCC and gene length for each Sample;
Supplementary files format and content: spermatogenesis_processed_file.xlsx:tab-delimited text files include normalized values by total reads and gene length for each Sample

value: adult mature sperm


title: 7E_M_689


title: 7E_M_688


title: 7E_M_687


title: N11N37 mature_sperm-1

description:
mature_sperm-1
spermatogenesis_processed_file.xlsx


title: 7E_M_686


title: N11N34 pachytene-2

description:
pachytene-2
spermatogenesis_processed_file.xlsx

value: adult pachytene


title: 7E_M_685


title: N11N33 pachytene-1

description:
pachytene-1
spermatogenesis_processed_file.xlsx

value: testes


title: 7E_M_684


title: 7E_M_682


title: N11N31 zygotene-1

description:
zygotene-1
spermatogenesis_processed_file.xlsx

value: male

value: adult zygotene


title: 7E_M_683


title: N11N30 leptotene-2

description:
leptotene-2
spermatogenesis_processed_file.xlsx

value: adult leptotene

value: wild type


title: 7E_M_681


title: 7E_M_691


title: 7E_M_680


title: 7E_M_679


title: N11N29 leptotene-1

description:
leptotene-1
spermatogenesis_processed_file.xlsx

value: FACS


title: 7E_M_678


title: N11N28 pre_leptotene-2

description:
pre_leptotene-2
spermatogenesis_processed_file.xlsx

value: adult pre-leptotene


title: N11N26 Spermatogonia-2

description:
Spermatogonia-2
spermatogenesis_processed_file.xlsx

value: adult spermatogonia


title: N11N27 pre_leptotene-1

description:
pre_leptotene-1
spermatogenesis_processed_file.xlsx


title: 7E_M_677


title: 7E_M_676


title: N11N22 Round_sperm-2

description:
Round_sperm-2
spermatogenesis_processed_file.xlsx

value: adult round sperm


title: F2R26 WT-3d-P4IP-2

description:
WT-3d-P4IP-2
smRNA_piRNA_file.zip


title: N11N21 Round_sperm-1

description:
Round_sperm-1
spermatogenesis_processed_file.xlsx


title: 7E_M_675


title: SF1R18 mature_sperm-2

description:
mature_sperm-2
smRNA_piRNA_file.zip


title: 7E_M_674


title: SF1R17 mature_sperm-1

description:
mature_sperm-1
smRNA_piRNA_file.zip


title: SF1R2 Round_sperm-2

description:
Round_sperm-2
smRNA_piRNA_file.zip


title: SF1R1 Round_sperm-1

description:
Round_sperm-1
smRNA_piRNA_file.zip


title: 7E_M_673


title: 7E_M_672


title: N11N32 zygotene-2

description:
zygotene-2
spermatogenesis_processed_file.xlsx


title: SF1R14 pachytene-2

description:
pachytene-2
smRNA_piRNA_file.zip


title: 7E_M_671


title: SF1R13 pachytene-1

description:
pachytene-1
smRNA_piRNA_file.zip


title: SF1R12 zygotene-2

description:
zygotene-2
smRNA_piRNA_file.zip


title: 7E_M_670


title: 7E_M_669


title: SF1R11 zygotene-1

description:
zygotene-1
smRNA_piRNA_file.zip


title: 7E_M_668


title: 7E_M_667


title: SF1R10 leptotene-2

description:
leptotene-2
smRNA_piRNA_file.zip


title: SF1R9 leptotene-1

description:
leptotene-1
smRNA_piRNA_file.zip


title: SF1R7 pre-leptotene-1

description:
pre-leptotene-1
smRNA_piRNA_file.zip


title: 7E_M_665


title: N11N25 Spermatogonia-1

description:
Spermatogonia-1
spermatogenesis_processed_file.xlsx


title: 7E_M_663


title: 7E_M_662


title: SF1R5 Spermatogonia-1

description:
Spermatogonia-1
smRNA_piRNA_file.zip


title: N12N20 P4KO-ad-2

description:
P4KO-ad-2
testis_processed_file.xlsx

value: adult


title: N12N19 P4KO-ad-1

description:
P4KO-ad-1
testis_processed_file.xlsx


title: 7E_M_666


title: 7E_M_661


title: N12N18 P3KO-ad-2

description:
P3KO-ad-2
testis_processed_file.xlsx


title: 7E_M_659


title: 7E_M_658


title: N12N14 P3KO-ad-1

description:
P3KO-ad-1
testis_processed_file.xlsx


title: 7E_M_664


title: 7E_M_660


title: 7E_M_657


title: N12N12 P2KO-ad-1

description:
P2KO-ad-1
testis_processed_file.xlsx


title: N12N13 P2KO-ad-2

description:
P2KO-ad-2
testis_processed_file.xlsx


title: N12N10 P1KO-ad-1

description:
P1KO-ad-1
testis_processed_file.xlsx


title: 7E_M_656


title: 7E_M_655


title: N12N11 P1KO-ad-2

description:
P1KO-ad-2
testis_processed_file.xlsx


title: SF1R8 pre-leptotene-2

description:
pre-leptotene-2
smRNA_piRNA_file.zip


title: 7E_M_652


title: 7E_M_654


title: N12N09 WT-ad-2

description:
WT-ad-2
testis_processed_file.xlsx


title: N12N08 WT-ad-1

description:
WT-ad-1
testis_processed_file.xlsx


title: N12N07 P4KO-3d-2

description:
P4KO-3d-2
testis_processed_file.xlsx

value: piwil4 -/-


title: SF1R6 Spermatogonia-2

description:
Spermatogonia-2
smRNA_piRNA_file.zip


title: 7E_M_651


title: N12N06 P4KO-3d-1

description:
P4KO-3d-1
testis_processed_file.xlsx


title: N12N05 P2KO-3d-2

description:
P2KO-3d-2
testis_processed_file.xlsx


title: 7E_M_650


title: 7E_M_648


title: N12N04 P2KO-3d-1

description:
P2KO-3d-1
testis_processed_file.xlsx


title: N12N03 WT-3d-2

description:
WT-3d-2
testis_processed_file.xlsx


title: 7E_M_649


title: 7E_M_647


title: N12N02 WT-3d-1

description:
WT-3d-1
testis_processed_file.xlsx


title: F2R16 P4KO-ad-2

description:
P4KO-ad-2
smRNA_piRNA_file.zip


title: F2R15 P4KO-ad-1

description:
P4KO-ad-1
smRNA_piRNA_file.zip


title: 7E_M_646


title: 7E_M_644


title: 7E_M_643


title: F2R13 P3KO-ad-1

description:
P3KO-ad-1
smRNA_piRNA_file.zip

value: piwil3 -/-


title: F2R14 P3KO-ad-2

description:
P3KO-ad-2
smRNA_piRNA_file.zip


title: 7E_M_653


title: F2R12 P2KO-ad-2

description:
P2KO-ad-2
smRNA_piRNA_file.zip

value: piwil2 -/-


title: Bone_old_CITE

city: Rochester

first: Madison

last: Doolittle

state: MN

address: 200 1st St SW

institute: Mayo Clinic

description:
Antibody-derived oligonucleotide (ADT)
old_cite_barcodes.tsv.gz
old_cite_features.tsv.gz
old_cite_matrix.mtx.gz

library_source: other

data_processing:
10X Genomics Cell Ranger Single Cell Software Suite (v6.0.0) was used to demultiplex raw base call (BCL) files generated from the sequencer into FASTQ files. The pipeline input FASTQ files for each sample to perform alignment to the reference genome, filtering, barcode counting and UMI counting. Further analyses were carried out in R version 4.0.3 using the Seurat package (v4.0)
Any remaining cells expressing markers for hematopoietic cells (Ptprc, Gypa, Tfrc, Cd3e, Cd19) were removed before analysis using the subset function in Seurat
Assembly: Genome_build: mm10
Supplementary files format and content: Tab-separated values files and matrix files
Library strategy: CITE-seq

molecule: protein

source_name: femur/tibia

extract_protocol:
Mice were euthanized according to standardized and approved IACUC protocols. Femurs and tibiae were isolated, cleaned of soft tissue, cut at both ends, and marrow centrifuged out of the diaphyses and metaphyses into a collection tube. Marrow was resuspended in 1mg/mL Liberase DL (Sigma) diluted in FACS buffer (0.5% BSA [Sigma] in PBS) and digested at 37°C for 30 minutes to increase yield of stromal cells released from the vasculature fraction. Diaphyses and metaphyses cleared of bone marrow were gently crushed, rinsed in PBS, and then digested in 300 Units/mL of Collagenase IA (Sigma), diluted in MEM α (ThermoFisher), 3 times for 25 minutes each. Bone and marrow solutions were then combined and treated with RBC lysis buffer (ThermoFisher) to clear erythrocytes. The sample was then depleted of cells expressing hematopoietic lineage markers (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119) using Magnet Assisted Cell Sorting (MACS) and the Lineage Cell Depletion Kit (Miltenyl Biotec). Samples were incubated with anti-mouse CD45-FITC at 1:400 dilution and anti-mouse CD24-TotalSeq B at 1:400 dilution in FACS buffer at 4°C for 20 minutes in the dark. Cells were then incubated with SYTOX blue at 1:4,000 for 5 minutes, spun down at 300xg for 5 minutes at 4°C, then resuspended in FACS buffer at 1x10^7 cells/mL and sorted for CD45-SyTOX- cells on a FACS Aria II (BD Biosciences). Live Lin-CD45- cells were then washed twice in 1x PBS + 0.04% BSA and immediately submitted to the Mayo Clinic Genome Analysis Core for single-cell sequencing.
Cells were counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter).  The Chromium Next GEM Single Cell 3’ Library and Gel Bead Kit (10x Genomics) was used for cDNA synthesis and standard Illumina sequencing primers and a set of unique i7 Sample dual indices (10x Genomics) were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific) and Agilent Bioanalyzer High Sensitivity chips (Agilent). Pooled libraries were then sequenced on Illumina NextSeq 2000 at 40,000 and 50,000 fragment reads/sample following Illumina’s standard protocol on a P2 flow cell. The P2 flow cell was sequenced at 100 x 2 paired end reads using a NextSeq P2 sequencing kit, NextSeq Control Software v1.4.1 and base-calling was analyzed using Illumina’s RTA version 3.4.4.

value: femur/tibia

tag: library type

value: ADT

tag: adt

value: TotalSeq™-B0212 anti-mouse CD24 Antibody

tag: cell type

value: Mesenchymal cells (CD45-Lin-)

value: INK-ATTAC


title: Bone_old_scRNA

description:
polyA RNA
old_cite_barcodes.tsv.gz
old_cite_features.tsv.gz
old_cite_matrix.mtx.gz

tag: tissue

value: mRNA


title: Bone_mesenchymal_old_rep2

data_processing:
10X Genomics Cell Ranger Single Cell Software Suite (v6.0.0) was used to demultiplex raw base call (BCL) files generated from the sequencer into FASTQ files. The pipeline input FASTQ files for each sample to perform alignment to the reference genome, filtering, barcode counting and UMI counting. Further analyses were carried out in R version 4.0.3 using the Seurat package (v4.0)
Any remaining cells expressing markers for hematopoietic cells (Ptprc, Gypa, Tfrc, Cd3e, Cd19) were removed before analysis using the subset function in Seurat
Assembly: Genome_build: mm10
Supplementary files format and content: Tab-separated values files and matrix files

extract_protocol:
Mice were euthanized according to standardized and approved IACUC protocols. Femurs and tibiae were isolated, cleaned of soft tissue, cut at both ends, and marrow centrifuged out of the diaphyses and metaphyses into a collection tube. Marrow was resuspended in 1mg/mL Liberase DL (Sigma) diluted in FACS buffer (0.5% BSA [Sigma] in PBS) and digested at 37°C for 30 minutes to increase yield of stromal cells released from the vasculature fraction. Diaphyses and metaphyses cleared of bone marrow were gently crushed, rinsed in PBS, and then digested in 300 Units/mL of Collagenase IA (Sigma), diluted in MEM α (ThermoFisher), 3 times for 25 minutes each. Bone and marrow solutions were then combined and treated with RBC lysis buffer (ThermoFisher) to clear erythrocytes. The sample was then depleted of cells expressing hematopoietic lineage markers (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119) using Magnet Assisted Cell Sorting (MACS) and the Lineage Cell Depletion Kit (Miltenyl Biotec). Samples were incubated with anti-mouse CD45-FITC at 1:400 dilution in FACS buffer at 4°C for 20 minutes in the dark. Cells were then incubated with SYTOX blue at 1:4,000 for 5 minutes, spun down at 300xg for 5 minutes at 4°C, then resuspended in FACS buffer at 1x10^7 cells/mL and sorted for CD45-SyTOX- cells on a FACS Aria II (BD Biosciences). Samples were then pooled, and live Lin-CD45- cells were washed twice in 1x PBS + 0.04% BSA and immediately submitted to the Mayo Clinic Genome Analysis Core for single-cell sequencing.
Cells were counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter).  The Chromium Next GEM Single Cell 3’ Library and Gel Bead Kit (10x Genomics) was used for cDNA synthesis and standard Illumina sequencing primers and a set of unique i7 Sample dual indices (10x Genomics) were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific) and Agilent Bioanalyzer High Sensitivity chips (Agilent). Libraries were sequenced at 50,000 fragment reads per cell following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit.  The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software.  Base-calling was performed using Illumina’s RTA version 2.7.3.

value: male


title: F2R11 P2KO-ad-1

description:
P2KO-ad-1
smRNA_piRNA_file.zip

value: testes


title: Bone_young_CITE

description:
Antibody-derived oligonucleotide (ADT)
young_cite_barcodes.tsv.gz
young_cite_features.tsv.gz
young_cite_matrix.mtx.gz


title: 7E_M_640


title: 7E_M_641


title: Bone_young_scRNA

description:
polyA RNA
young_cite_barcodes.tsv.gz
young_cite_features.tsv.gz
young_cite_matrix.mtx.gz


title: Bone_mesenchymal_old_rep1


title: F2R10 P1KO-ad-2

description:
P1KO-ad-2
smRNA_piRNA_file.zip

value: piwil1 -/-


title: 7E_M_639


title: Untagged_IAA_RNAseq_Rep3

city: Pittsburgh

middle: Charles

last: Klein

address: 4249 Fifth Ave

department: Biological Sciences

institute: University of Pittsburgh

data_processing:
Barcode adapter sequences removed
Reads mapped to mm10 genome using STAR, with options --outFilterMismatchNoverReadLmax 0.02 and --outFilterMultimapNmax 1
Aligned reads were processed in deepTools by using the bamCoverage command to generate stranded bigwig files detailing coverage genome-wide in 1 bp bins with BPM normalization
Assembly: mm10
Supplementary files format and content: bigwig

source_name: cell line

growth_protocol: Murine embryonic stem cells were grown under feeder-free conditions in DMEM, supplemented with 10% Fetal Bovine Serum, 2 mM-Glutamine, 1X non-essential amino acids, 0.129mM 2-mercaptoethanol, 1000U/mL Leukemia Inhibitory Factor (LIF), 3 µM CHIR99021 GSK inhibitor (p212121), and 1 µM PD0325091 MEK inhibitor (p212121). Cells were passaged every 48 hours using trypsin (Gibco) and split at a ratio of ~1:8 with fresh medium. Routine anti-mycoplasma cleaning was conducted (LookOut DNA Erase spray, Sigma) and cell lines were screened by PCR to confirm no mycoplasma presence.

extract_protocol:
Medium was aspirated and cells were washed with DPBS, then trypsinized. Trypsinization reaction was quenched with untreated medium. Cells were pelleted, washed with DPBS, and RNA was extracted with TRIzol per manufacturer's instructions.
RNA-seq; built with NEBNext Directional kit

treatment_protocol: After 48 hours of undisturbed growth on 10 cm plates, medium was aspirated and cells were washed with DPBS. DPBS was aspirated and fresh medium was added containing 500 nM 3-Indoleacetic acid (3-IAA) or an equivalent volume of 100% ethanol. Plates were then left at 37 degrees Celsius with 5% CO2 for 24 hours before extraction.

value: cell line

tag: cell line

value: murine embryonic stem cells

value: IAA

value: Cells were left undisturbed on 10 cm plates for 48 hours prior to treatment. Cells were treated with 0.1% ethanol mixed into medium for the indicated time (hours). Cells were incubated at 37 degrees Celsius with 5% CO2 for the duration of treatment.


title: Untagged_IAA_RNAseq_Rep2


title: Untagged_IAA_RNAseq_Rep1


title: SPT16_V5_AID_Clone2_IAA_RNAseq_Rep1


title: 7E_M_645


title: F2R08 WT-ad-2

description:
WT-ad-2
smRNA_piRNA_file.zip

tag: Sex

value: adult

value: wild type


title: SPT16_V5_AID_Clone2_IAA_RNAseq_Rep2


title: 7E_M_510


title: F2R09 P1KO-ad-1

description:
P1KO-ad-1
smRNA_piRNA_file.zip

tag: genotype


title: 7E_M_642


title: F2R07 WT-ad-1

description:
WT-ad-1
smRNA_piRNA_file.zip


title: 7E_M_509


title: SPT16_V5_AID_Clone2_EtOH_RNAseq_Rep2

tag: treatment

value: EtOH


title: SPT16_V5_AID_Clone1_IAA_RNAseq_Rep2


title: SPT16_V5_AID_Clone1_IAA_RNAseq_Rep1


title: F2R06 P4KO-3d-2

description:
P4KO-3d-2
smRNA_piRNA_file.zip

value: piwil4 -/-


title: F2R05 P4KO-3d-1

description:
P4KO-3d-1
smRNA_piRNA_file.zip


title: SPT16_V5_AID_Clone1_EtOH_RNAseq_Rep2


title: SPT16_V5_AID_Clone1_EtOH_RNAseq_Rep1


title: 7E_M_508

tag: brain region


title: SPT16_AID_V5_Clone2_IAA_TTseq_washout_Rep1

description: Nascent RNA

data_processing:
library strategy: TT-seq
Barcode adapter sequences removed
Reads mapped to mm10 genome using STAR, with options --outFilterMismatchNoverReadLmax 0.02 and --outFilterMultimapNmax 1
Aligned reads were processed in deepTools by using the bamCoverage command to generate stranded bigwig files detailing coverage genome-wide in 1 bp bins with BPM normalization
Assembly: mm10
Supplementary files format and content: bigwig

extract_protocol:
Medium was aspirated and cells were washed with DPBS. DPBS was aspirated and replaced with fresh medium containing 500 mM 4-thiouridine (4sU). Cells were incubated at 37 degrees Celsius with 5% CO2 for five minutes, then washed with DPBS and trypsinized. Cells were pelleted, washed with DPBS, and RNA was extracted with TRIzol per manufacturer's instructions.
TT-seq; built with NEBNext Directional kit

treatment_protocol: After 48 hours of undisturbed growth on 10 cm plates, medium was aspirated and cells were washed with DPBS. DPBS was aspirated and fresh medium was added containing 500 nM 3-Indoleacetic acid (3-IAA) or an equivalent volume of 100% ethanol. Plates were then left at 37 degrees Celsius with 5% CO2 for 12 hours, then medium was replaced with fresh medium, and cells were incubated for 24 hours before extraction.


title: SPT16_AID_V5_Clone2_IAA_TTseq_washout_Rep2

description: Nascent RNA


title: 7E_M_507

tag: age


title: F2R04 P2KO-3d-2

description:
P2KO-3d-2
smRNA_piRNA_file.zip

value: piwil2 -/-


title: F2R03 P2KO-3d-1

description:
P2KO-3d-1
smRNA_piRNA_file.zip


title: 7E_M_506

tag: strain


title: SPT16_AID_V5_Clone2_EtOH_TTseq_washout_Rep2

description: Nascent RNA


title: 7E_M_638


title: SPT16_AID_V5_Clone2_EtOH_TTseq_washout_Rep1

description: Nascent RNA


title: SPT16_AID_V5_Clone1_IAA_TTseq_washout_Rep2

description: Nascent RNA


title: SPT16_V5_AID_Clone2_EtOH_RNAseq_Rep1


title: 7E_M_505


title: SPT16_AID_V5_Clone1_EtOH_TTseq_washout_Rep2

description: Nascent RNA


title: 7E_M_504


title: F2R02 WT-3d-2

description:
WT-3d-2
smRNA_piRNA_file.zip


title: 7E_M_503


title: SPT16_AID_V5_Clone1_EtOH_TTseq_washout_Rep1

description: Nascent RNA


title: SPT16_AID_V5_Clone2_IAA_ATAC_washout_Rep2

data_processing:
Reads were trimmed to 25 bp and barcode adapter sequences removed using trim_galore
Reads mapped to mm10 genome using bowtie2, allowing for 1 mismatch
Duplicates were removed using Picard
Reads were filtered for quality using SAMtools (MAPQ ≥ 10) and size-selected into nucleosome-free (1-100 bp) size class
Reads were processed in deepTools by using the bamCoverage command to generate bigwig files detailing coverage genome-wide in 1 bp bins with RPGC normalization, using an effectiveGenomeSize of 2308125349
Assembly: mm10
Supplementary files format and content: bigwig

extract_protocol:
Medium was aspirated and cells were washed with DPBS. DPBS was aspirated and cells were trypsinized. Cells were pelleted, washed with DPBS, and resuspended in hypotonic buffer. Nuclei were pelleted and lysed cell debris (supernatant) removed. Nuclei were flash-frozen until time of use. Nuclei were transposed for 30 minutes at 37C with 100 rpm shaking and Tn5 transposome (Diagenode). Libraries were amplified for 10 cycles of high-fidelity PCR
Transposed for 30 minutes at 37C with 100 rpm shaking and Tn5 transposome (Diagenode). Libraries were amplified for 10 cycles of high-fidelity PCR

treatment_protocol: After 48 hours of undisturbed growth on 10 cm plates, medium was aspirated and cells were washed with DPBS. DPBS was aspirated and fresh medium was added containing 500 nM 3-Indoleacetic acid (3-IAA) or an equivalent volume of 100% ethanol. Plates were then left at 37 degrees Celsius with 5% CO2 for 12 hours, then incubated in fresh medium without treatment for 24 hours before extraction.


title: SPT16_AID_V5_Clone2_IAA_ATAC_washout_Rep1


title: SPT16_AID_V5_Clone2_EtOH_ATAC_washout_Rep1


title: N10N64 P3KO-54PEA-2

description:
P3KO-54PEA-2
oocyte_processed_file.xlsx

source_name: embryo

value: embryo

value: female

value: piwil3 -/-


title: N10N66 P3KO-54PEA-4

description:
P3KO-54PEA-4
oocyte_processed_file.xlsx


title: 7E_M_502


title: SPT16_AID_V5_Clone2_EtOH_ATAC_washout_Rep2


title: N10N62 P3KO-34PEA-5

description:
P3KO-34PEA-5
oocyte_processed_file.xlsx


title: SPT16_AID_V5_Clone1_IAA_ATAC_washout_Rep2


title: 7E_M_501


title: SPT16_AID_V5_Clone1_EtOH_ATAC_washout_Rep2


title: N10N57 P3KO-34PEA-3

description:
P3KO-34PEA-3
oocyte_processed_file.xlsx


title: 7E_M_499


title: SPT16_AID_V5_Clone1_EtOH_ATAC_washout_Rep1


title: N10N55 P3KO-34PEA-1

description:
P3KO-34PEA-1
oocyte_processed_file.xlsx


title: Mouse 2 scRNA

city: Münster

first: Carolin

last: Walter

address: Domagkstraße 9

department: Medical Faculty of the WWU Münster

library_source: transcriptomic single cell

data_processing:
All samples were demultiplexed and then processed with the 10x Genomics CellRanger v6.0.2 pipeline.
For all samples, the raw 10X scRNA-seq data was converted to fastq format with CellRanger's mkfastq function.
CellRanger's count algorithm was then used to align the data against the murine reference transcriptome mm10, version 2020-A.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

molecule: polyA RNA

source_name: tumor

extract_protocol:
The two tumors were isolated and minced with scalpels on ice. For enzymatic dissociation, 5 ml Papain solution (in DMEM/F12) supplmented with 1000 U DNase ( was added to each sample, followed by incubation at 37°C in 5% CO2 for 30 min. The cell solution was then transferred to a 40 µm cell strainer. Red blood cells were lysed with ACK lysis buffer (5 min. at 4°C). The resulting single cell suspensions were stained with 7-aminoactin mycin D (7-AAD). Non-viable, 7-AAD-positive cells were removed by fluorescence-activated cell sorting before viable single cells were further processed using 10x Genomics Chromium Next GEM Single Cell 3' Technology.
Approximately 10,000 single cells of each sample were processed for scRNA-seq using the Chromium Next GEM Single Cell 3’ technology (10X Genomics) according to the manufacturer's instructions. In brief, single-cell GEMs (gel beads in emulsion) were generated by the Chromium Controller, followed by GEM reverse transcription, a cleanup using Dynabeads, cDNA amplification, cleanup and size selection using SPRIselect beads, and construction of the sequencing library using Library & Gel Bead Kit v3.1, the Chromium Next GEM Chip G and the Single Index Kit T. Quality, purity, average size and concentration of cDNA and libraries were determined by measurements on a Tapestation 2200 (Agilent Technologies). Indexed libraries were sequenced using an Illumina NextSeq 2000 sequencing platform.

value: tumor

value: 100 days


title: 7E_M_500


title: F2R01 WT-3d-1

description:
WT-3d-1
smRNA_piRNA_file.zip

value: male


title: SPT16_AID_V5_Clone1_IAA_TTseq_washout_Rep1

description: Nascent RNA


title: 7E_M_498


title: 7E_M_497


title: N10N54 P3KO-11PEA-5

description:
P3KO-11PEA-5
oocyte_processed_file.xlsx


title: Mouse 1 scRNA

value: 90 days


title: N10N53 P3KO-11PEA-4

description:
P3KO-11PEA-4
oocyte_processed_file.xlsx


title: PBMC_K4me123_B_1X_(220826_DJ_Hs_PBMC_K4me123_MM_1X_220527)

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

description: sciCUT&Tag for H3K4me123 run on PBMCs from healthy donor B and dispensed on the ICELL8 at a concentration of 24 nuclei per nanowell

data_processing:
Genome_build: hg38
1. We used cutadapt 2.9 with parameters "-j 8 --nextseq-trim 20 -m 20 -a  AGATCGGAAGAGCACACGTCTGAACTCCAGTCA -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -Z" to trim adapters from 50bp paired-end reads. 2. We used Bowtie2 2.4.2 with options "--very-sensitive-local --soft-clipped-unmapped-tlen --dovetail --no-mixed --no-discordant -q --phred33 -I 10 -X 1000" to map the paired-end 50bp reads to the repeat-masked hg38 Homo sapiens reference sequence obtained from UCSC. 3. We used samtools 1.14 "view" to extract properly paired reads from the D. melanogaster alignments. 4. We used bedtools 2.30.0 "genomecov" to make a normalized count hg38 track which is the fraction of counts at each base pair scaled by the size of the hg38 reference sequence (3210038034) so that if the counts were uniformly distributed across the reference sequence there would be one at each position (Supplementary file .bw)

source_name: sciCUT&Tag

tag: scicut&tag antibody

value: A mixture of H3K4me1 Invitrogen cat# 710795, H3K4me2 Millipore cat# 07-030, H3K4me3 Active Motif cat# 39159

tag: human sample

value: PBMCsw from healthy donor B


title: PBMC_K4me123_B_PE79_(220901_DJ_Hs_PBMC_K4me123_MM_PE79_220523)

description: sciCUT&Tag for H3K4me123 run on PBMCs from healthy donor B


title: PBMC_K4me123_B_0.25X_(220826_DJ_Hs_PBMC_K4me123_MM_0.25X_220527)

description: sciCUT&Tag for H3K4me123 run on PBMCs from healthy donor B and dispensed on the ICELL8 at a concentration of 6 nuclei per nanowell


title: PBMC_K4me123_B_0.5X_(220826_DJ_Hs_PBMC_K4me123_MM_0.5X_220527)

description: sciCUT&Tag for H3K4me123 run on PBMCs from healthy donor B and dispensed on the ICELL8 at a concentration of 12 nuclei per nanowell


title: N10N52 P3KO-11PEA-3

description:
P3KO-11PEA-3
oocyte_processed_file.xlsx


title: PBMC_K4me123_AnB_PE79_(220901_DJ_Hs_PBMC_K4me123_DJnMM_PE79_220523)

description: sciCUT&Tag for H3K4me123 run on a mixture of PBMCs from healthy donor A and donor B


title: 7E_M_495


title: N10N51 P3KO-11PEA-2

description:
P3KO-11PEA-2
oocyte_processed_file.xlsx


title: PBMC_K4me123_B_0.125X_(220826_DJ_Hs_PBMC_K4me123_MM_0.125X_220527)

description: sciCUT&Tag for H3K4me123 run on PBMCs from healthy donor B and dispensed on the ICELL8 at a concentration of 3 nuclei per nanowell


title: 7E_M_496


title: PBMC_K4me123_AnB_1X_(220826_DJ_Hs_PBMC_K4me123_DJnMM_1X_220527)

description: sciCUT&Tag for H3K4me123 run on a mixture of PBMCs from healthy donor A and donor B and dispensed on the ICELL8 at a concentration of 24 nuclei per nanowell


title: N10N50 P3KO-11PEA-1

description:
P3KO-11PEA-1
oocyte_processed_file.xlsx