title: GSM9162838: fetal brain, disomic, Library B15C1H_atac, Chromatin Accessibility, low quality/non-cortical, NOT IN FINAL ANALYSIS; Homo sapiens; ATAC-seq
library_construction_protocol: Fresh-frozen fetal tissue from surgical terminations of pregancy from UK hospitals was collected by the "Human Developmental Biology Resource" (HDBR, https://www.hdbr.org/). 10-50 mg of fetal tissue were processed using a protocol based on dx.doi.org/10.17504/protocols.io.bvhhn336. After removal from dry ice, tissue was immediately suspended in Homogenisation Buffer (10 mM Tris-HCl pH7.4, 320 mM Sucrose, 3 mM CaCl2, 3 mM MgCl2), supplemented freshly with 0.1% NP-40, 1 mM DTT and 1 U/µl RNAse inhibitor (Protector (Sigma #3335402001), RiboLock (Thermo Fisher, PN-EO0382), or RNaseOUT (Thermo Fisher, 10777019) RNase inhibitor). The tissue was then immediately homogenised using a 1-ml dounce homogeniser and exactly after 5 min diluted with 1 volume of Homogenisation Buffer (without NP-40), filtered (30 µm mesh size) to remove large debris, and centrifuged (500g, 5 min, 4°C) to collect raw nuclei. Raw nuclei were resuspended in Homogenisation Buffer (without NP-40) and mixed with 1 volume 50% iodixanol buffer (10 mM Tris-HCl pH7.4, 3 mM CaCl2, 3 mM MgCl2, 1 mM DTT, 0.5 U/µl RNAse inhibitor). This suspension was carefully overlaid on a 29% iodixanol buffer (as above + 160 mM sucrose), and centrifuged (6000g, 30 min, 4°C). The nuclei pellet was resuspended in a Lysis Buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1 mM DTT, 0.5 U/µl RNAse inhibitor, 0.1% Tween-20, 0.1% NP-40, 0.001% digitonin), and after exactly 2 min nuclei were diluted in 10 volumes wash buffer (lysis buffer without NP-40, digitonin), and centrifuged (500g, 5 min, 4°C) to collect the nuclei, which were then resuspended in 1x Nuclei Buffer (from the “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle”; 10X Genomics PN-1000283/PN-1000285; supplemented with 1 mM DTT, 0.5 U/µl RNAse inhibitor). snRNA-seq and snATAC-seq libraries were prepared using a 10X Genomics Chromium X and the “Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle” (10X Genomics PN-1000283/PN-1000285) according to manufacturer's instructions. Libraries were sequenced using Illumina Nextseq2000 or Novaseq6000 sequencers.