title: GSM3691034: 2CSE-113 [2 cell stage]; Mus musculus; OTHER
library_construction_protocol: ]following fixation, and brief rinses in 1X PBS solution 1% acetylated BSA (Sigma), blastomeres were permeabilized for 30 min on ice in 10 mM Tris-HCl (pH 8), 10 mM NaCl, 0. 2% IGEPAL CA-630 containing complete EDTA-free protease inhibitor cocktail (Roche). Cells were transferred to a protein Low Binding tube (Sigma) containing 0. 3% SDS diluted with 1. 24 x NEBuffer3 for 60 min at 37 ̊C with constant agitation. Triton X-100 was added to 2% final and incubation was extended for 60 min, before addition of 625U of Mbo I (New England Biolabs) and overnight incubation. To label the digested DNA ends, 28. 4uM final of dC/G/TPs and biotin-14 dATP were added with 25U DNA polymerase I, large (Klenow) fragment (New England Biolabs) for 60 min with constant agitation. After spinning, blastomeres were treated with 10 U of T4 DNA ligase (Thermo Fisher) in presence of 1X reaction buffer with 1XBSA ( both by New England Biolabs) at 16 ̊C for at least 4 hours. After spinning, blastomeres were resuspended with PBS 1x and BSA 1 mg/mL to dispatch them individually into PCR tubes (in strips; one per tube) before storage at -80 ̊C until further processing. To prepare single-cell Hi-C libraries from single nuclei in PCR strips, 5 μl of PBS was added to each well and crosslinks reversed by incubating at 65 ̊C overnight. Hi-C concatemer DNA was fragmented and linked with sequencing adapters using the Nextera XT DNA Library Preparation Kit (Illumina), by adding 10 μl of Tagment DNA Buffer and 5 μl of Amplicon Tagment Mix, incubating at 55 ̊C for 20 min, then cooling down to 10 ̊C, followed by addition 5 μl of Neutralize Tagment Buffer and incubation for 5 min at room temperature. Hi-C ligation junctions were then captured by Dynabeads M-280 streptavidin beads (Thermo Fisher; 20 μl of original suspension per single-cell sample). Beads were prepared by washing with 1 x BW buffer (5 mM Tris-Cl pH 7. 5, 0. 5 mM EDTA, 1 M NaCl), resuspended in 4 x BW buffer (20 mM Tris-Cl pH 7. 5, 2 mM EDTA, 4 M NaCl; 8 μl per sample), and then mixed with the 25 μl sample and incubated at room temperature overnight with gentle agitation. The beads were then washed four times with 200 μl of 1 x BW buffer, twice with 200 μl of 10 mM Tris-Cl pH 7. 5 at room temperature, and resuspended in 25 μl of 10 mM Tris-Cl pH 7. 5. Single-cell Hi-C libraries were amplified from the beads by adding 15 μl of Nextera PCR Master Mix, 5 μl of i7 Index primer of choice and 5 μl of i5 Index primer of choice. Samples were then incubated at 72 ̊C for 3 min, 95 ̊C for 30 sec followed by the thermal cycling at 95 ̊C for 10 sec, 55 ̊C for 30 sec and 72 ̊C for 30 sec for 18 cycles, then incubated at 72 ̊C for 5 min. The supernatant was separated from the beads and purified one by one with AMPure XP beads (Beckman Coulter; 0. 6 times volume of the supernatant) according to manufacturer's instructions and eluted with 30 μl each of 10 mM Tris-Cl pH 8. 5. The eluate was purified once more with AMPure XP beads (equal volume to the previous eluate) and eluted with 11 μl of 10 mM Tris-Cl pH 8. 5.
attributes.tag: GEO Accession
attributes.value: GSM3691034