Electron tomograms from lamina-associated heterochromatin upon stretch

Data includes ET tilt series and reconstructions from samples prepared on 1.2.2029 and 9.10.2019. Sara Wickström, Kate Miroshnikova Primary human epidermal progenitor cells were grown on elastomere membranes exposed for 0 min, 30 min, 180 min or 360 min 40% stretch. Samples were fixed with 2.5% GA in 5 mM CaCl2, 0.1 M sodium cacodylate buffer, pH 7.4 at RT for 5 min, following 1 h fixation on ice. Following fixation all the steps were performed on ice. Samples were first washed 5 times with 0.1 M sodium cacodylate buffer and blocked for 15 min in 10 mM glycine/10 mM potassium cyanide in 0.1 M sodium cacodylate buffer (blocking buffer). Following blocking samples were rinsed 1X with 0.1 M sodium cacodylate buffer and then stained with 10 μM DRAQ5 in 0.1% saponin in 0.1 M sodium cacodylate buffer for 10 min, followed by washing 3x for 5 min with above-described blocking buffer. Samples were then incubated with 2.5 mM diaminobezidine tetrahydrochloride in 0.1 M sodium cacodylate buffer and placed onto a glass-bottom dish, monolayer side down, then mounted onto Zeiss confocal microscope surrounded by ice-packs to maintain a near 4°C temperature and imaged using a 63X oil immersion objective lens and 633 nm filter set to continuously illuminate the membranes for 10 min. Samples were then rinsed 3x for 5 min with 0.1 M sodium cacodylate buffer, following a 1 h stain with 1% osmium tetroxide, 2 mM CaCl2, 1.5% potassium ferrocyanide in 0.15 M sodium cacodylate buffer. Post staining, samples were washed twice for 2 min with double-distilled water, dehydrated in ethanol series prior to gradual infiltration into epoxy (TAAB 812). After polymerization in Epon overnight at 60°C, a pyramid was made using a razor blade on the area of cells of darker, photo-oxidized nucleus. 230-nm-thick sections were cut with a 35° diamond knife and collected on Pioloform-coated single slot copper grids. Dual axis tilt series were recorded from a semi-thick section using SerialEM software running on a Tecnai FEG 20 TEM operated at 200 kV, high tilt specimen holder and a 4k by 4k Ultrascan CCD camera. The tilt series were acquired at one-degree intervals between ± 62° at nominal magnification of 14,500x and 10-nm gold particles placed on both grid faces served as fiducial markers for alignment. Prior alignment and reconstruction with IMOD software package the images were binned by two providing a pixel size of 1.5 nm. The tomograms were reconstructed with a simultaneous iterative reconstruction technique using 14 iterations. Nava MM, Miroshnikova YA, Biggs LC, Whitefield DB, Metge F, Boucas J, Vihinen H, Jokitalo E, Li X, García Arcos JM, Hoffmann B, Merkel R, Niessen CM, Dahl KN, Wickström SA. Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage. Cell. 2020 May 14;181(4):800-817.e22. doi: 10.1016/j.cell.2020.03.052. Epub 2020 Apr 16. PMID: 32302590; PMCID: PMC7237863. Fig S3E, videos S1, S2 and S3.

本数据集包含2029年2月1日与2019年10月9日制备的样品的电子断层扫描（Electron Tomography, ET）倾斜序列及重建结果，相关实验由Sara Wickström与Kate Miroshnikova开展。
原代人表皮祖细胞接种于弹性体膜上，分别施加40%的拉伸应力并持续0 min、30 min、180 min或360 min。
样品先用含2.5%戊二醛（Glutaraldehyde, GA）、5 mM氯化钙、0.1 M二甲胂酸钠缓冲液（pH 7.4）的溶液在室温下固定5 min，随后置于冰上继续固定1 h。固定完成后所有操作均在冰上进行。
样品先用0.1 M二甲胂酸钠缓冲液洗涤5次，随后置于含10 mM甘氨酸/10 mM氰化钾的0.1 M二甲胂酸钠缓冲液（封闭缓冲液）中封闭15 min。封闭完成后，样品用0.1 M二甲胂酸钠缓冲液漂洗1次，再用含10 μM DRAQ5、0.1%皂苷的0.1 M二甲胂酸钠缓冲液染色10 min，最后用上述封闭缓冲液洗涤3次，每次5 min。
随后将样品置于含2.5 mM二氨基联苯胺四盐酸盐（Diaminobenzidine tetrahydrochloride, DAB）的0.1 M二甲胂酸钠缓冲液中孵育，将细胞单层朝下放置于玻璃底培养皿中，随后转移至环绕冰袋以维持近4℃温度的蔡司（Zeiss）共聚焦显微镜载物台，使用63×油浸物镜与633 nm滤光组对膜结构进行连续10 min的照明成像。
成像完成后，样品用0.1 M二甲胂酸钠缓冲液漂洗3次，每次5 min；随后用含1%四氧化锇、2 mM氯化钙、1.5%铁氰化钾的0.15 M二甲胂酸钠缓冲液染色1 h。染色结束后，样品用双蒸水洗涤2次，每次2 min，经乙醇梯度脱水后逐步浸润至环氧树脂（TAAB 812）中。将样品置于60℃烘箱中聚合过夜（Epon包埋），随后用剃须刀片在光氧化致细胞核着色较深的细胞区域制作树脂金字塔块。
使用35°金刚石刀将包埋块切成230 nm厚的切片，并收集于涂有Pioloform的单缝铜网上。使用搭载SerialEM软件的Tecnai FEG 20透射电子显微镜（Transmission Electron Microscopy, TEM），于200 kV加速电压下，配合高倾斜样品台与4k×4k Ultrascan CCD相机，从半厚切片上采集双轴倾斜序列。该倾斜序列的采集范围为±62°，步长1°，标称放大倍数为14500×；铜网两面均放置10 nm金颗粒作为对齐fiducial标记物（fiducial marker）。在使用IMOD软件包进行对齐与重建前，将图像进行2倍合并，最终像素尺寸为1.5 nm。本研究采用同步迭代重建技术，经14次迭代完成断层图像重建。
本数据集关联引用文献如下：
Nava MM, Miroshnikova YA, Biggs LC, Whitefield DB, Metge F, Boucas J, Vihinen H, Jokitalo E, Li X, García Arcos JM, Hoffmann B, Merkel R, Niessen CM, Dahl KN, Wickström SA. 异染色质介导的细胞核软化保护基因组免受机械应力诱导损伤. Cell. 2020年5月14日;181(4):800-817.e22. DOI: 10.1016/j.cell.2020.03.052. 2020年4月16日在线发表. PMID: 32302590; PMCID: PMC7237863.
本数据集对应补充图S3E、视频S1、S2及S3。