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| Status |
Public on Nov 07, 2018 |
| Title |
SmcHD1_wt_ctr_rep2 |
| Sample type |
SRA |
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| Source name |
female clonal MEFs
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| Organism |
Mus musculus |
| Characteristics |
genetic background: Inter-specific: F2, M.m.domesticus FVB x M.m.Castaneus
genotype: wild-type
clone: Wt2A1
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| Growth protocol |
SmcHD1 WT, MommeD1 and CRISPR KO cell lines were grown on 5-8x145mm petri dishes in EC10 medium to semi-confluency.
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| Extracted molecule |
total RNA |
| Extraction protocol |
WT, SmcHD1 null and acute SmcHD1 null MEFs were grown on 2x145mm petri dishes in EC10 medium to semi-confluency. RNA-Seq enriching for chromatin-bound nuclear RNA was performed according to a modified chromatin RNA-Seq protocol 59 . The cells were trypsinised, collected in EC10 medium and cell number was counted on LUNAII cell counter (Logos Biosystems). 1x10^7 cells for each line were spun down, resuspended in 12ml of ice cold PBS and supplemented with 2.5x10^6 SG4 drosophila cells for calibration. Cells were spun down at 500g, 5 min at 4 0 C and cell pellets were resuspended in 800l of HLBN hypotonic buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl 2 , 0.05% NP40). 480 μl of buffer HLBNS (HLBN, 25% sucrose) was carefully under-layered to create sucrose cushion, and nuclei were isolated by centrifugation for 5 min at 1000g at 4 0 C. Supernatant containing cytoplasmic debris was discarded and the nuclear pellet was re-suspended in 100 μl of ice-cold buffer NUN1 (20 mM Tris-HCl pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol; 1 mM DTT and cOmplete EDTA free protease inhibitors (Sigma) added fresh). Nuclei were lysed in 1200 μl of ice-cold lysis buffer NUN2 (20 mM HEPES pH7.6, 300 mM NaCl, 7.5 mM MgCl 2 , 0.2 mM EDTA, 1 M urea, 1% NP40; 1 mM DTT) during 15min incubation on ice and RNA-bound chromatin was pelleted at 16000 g for 10min at 40C. Chromatin-RNA pellet was re-suspended in 200 μl of high salt buffer HSB (10 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM MgCl 2 ). DNA and proteins were digested with Turbo DNAse (Life Sciences) and proteinase K (10 mg/ml, ThermoFisher, nuclease free), incubating on ThermoMixer at 37 0 C for 10 min and 30min, respectively. RNA was extracted with 1 ml of TRIzol (Life Sciences) according to the manufacturer guidelines. RNA was dissolved in 1xTURBO DNAse buffer, digested with TURBO DNAse for 30 min at 37o C on a ThermoMixer and extracted with TRIzol. RNA was washed three times with 75% ethanol, dissolved in water and quantified using a NanoDrop (ND-1000). RNA quality was checked with RNA 6000 Pico Chip (Agilent Technologies) on Agilent 2100 Bioanalyser (Agilent Technologies). Samples were depleted of ribosomal RNA with Ribo-Zero Gold Kit (MRZG12324, Illumina) according to manufacturer’s guidelines. RNA was isolated from 3 biological replicates. RNA-Seq libraries were prepared with NEBNext® UltraTM Directional RNA Library Prep Kit for Illumina (E7420S) using NEBNext® Multiplex Oligos for Illumina for multiplexing (E7335S and E7500S). Libraries were sequenced on HiSeq2000 and NextSeq500 using NextSeq 500 High-Output Kit: 1 lane, 150 cycles, 75 bp paired end sequencing (Illumina).
NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
processed files were generated from merged bam files coming from all replicates and contain density plots (rpm, bigWigs) for both unsorted and allele-specific reads (FVB and M.m.castaneus) seperately fro each strand
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| Data processing |
Reads were mapped with STAR 2.5b aligner with index generated from FVB-Cast SNP N-masked genome and allele-specificaly sorted with SNPsplit.
For visualisation genome-wide coverage within 50 bp were calculated normalised for sequencing depth (rpm). BigWig files were generated to visualize the data in the UCSC browser.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig
created form bedGraph file (table: chr, start, end, value) with the bedGraphToBigWig utility available form UCSC; scores represent sequencing-depth normalized read density or log2(IP/input)
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| Submission date |
Oct 12, 2018 |
| Last update date |
Nov 07, 2018 |
| Contact name |
Michal R. Gdula |
| E-mail(s) |
michal.gdula@bioch.ox.ac.uk
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| Organization name |
Oxford University
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3RF |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE115984 |
The non-canonical SMC protein SmcHD1 antagonises TAD formation on the inactive X chromosome |
| GSE121184 |
chrRNA-seq in wild-type, SmcHD1 MommeD1mut and somKO MEFs |
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| Relations |
| BioSample |
SAMN10236570 |
| SRA |
SRX4873412 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3427472_Wt_repli_ctr_rep2_Cast_fwd.bw |
29.4 Mb |
(ftp)(http) |
BW |
| GSM3427472_Wt_repli_ctr_rep2_Cast_rev.bw |
30.5 Mb |
(ftp)(http) |
BW |
| GSM3427472_Wt_repli_ctr_rep2_FVB_fwd.bw |
113.0 Mb |
(ftp)(http) |
BW |
| GSM3427472_Wt_repli_ctr_rep2_FVB_rev.bw |
107.2 Mb |
(ftp)(http) |
BW |
| GSM3427472_Wt_repli_ctr_rep2_fwd.bw |
258.0 Mb |
(ftp)(http) |
BW |
| GSM3427472_Wt_repli_ctr_rep2_rev.bw |
249.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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