title: GSM3624706: mESC.DpnI.Dam_Ring1B.clone_18.Unsorted.differentiation_day3.32.library_KIN3147_11; Mus musculus; OTHER
library_construction_protocol: F1 mESCs expressing Dam-LMNB1, Dam or Dam-RING1B were collected in plain or 2i ES cell culture media and stained with 30 μg/mL Hoechst 34580 (Sigma, 63493) for 45 minutes at 37 °C. mESC singlets were sorted based on forward and side-scatter properties, and in mid-S phase of the cell cycle based on DNA content histogram. Differentiated F1 mESCs expressing Dam-RING1B were collected in differentiation media and stained with 30 μg/mL Hoechst 34580 for 45 minutes at 37 °C. The same cells were stained with 1 μg/mL propidium iodide (Sigma) for live/dead selection. Differentiated mESCs singlets were sorted based on forward and side-scatter properties, and in G1, S and G2/M phase of the cell cycle based on DNA content histogram. One cell was sorted per well of 384-well plates (Biorad, HSP3801) using the BD FACSJazz cell sorter. Wells contained 4 μL mineral oil (Sigma) and 100 nL of 15 ng/μL unique CELseq primer. 4 μL mineral oil was dispensed manually into each well of a 384-well plate using a multichannel pipet. 100 nL of unique CEL-Seq primer was dispensed per well using the mosquito HTS robot (TTP Labtech). The NanodropII robot (BioNex) was used for all subsequent dispensing steps at 12 p.s.i. pressure. After sorting, 100 nL lysis mix was added (0.8 U RNase inhibitor (Clontech, 2313A), 0.07 % Igepal, 1 mM dNTPs, 1:500,000 ERCC RNA spike-in mix (Ambion, 4456740)). Each single cell was lysed at 65 °C for 5 minutes and 150 nL reverse transcription mix was added (1x First Strand Buffer (Invitrogen, 18064-014), 10 mM DTT (Invitrogen, 18064-014), 2 U RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019), 10 U SuperscriptII (Invitrogen, 18064014)) and the plate was incubated at 42 °C for 1 hour, 4 °C for 5 minutes and 70 °C for 10 minutes. Next, 1.92 μL of second strand synthesis mix was added (1x second strand buffer (Invitrogen, 10812014), 192 μM dNTPs, 0.006 U E. coli DNA ligase (Invitrogen, 18052019), 0.013 U RNAseH (Invitrogen, 18021071)) and the plate was incubated at 16 °C for 2 hours. 500 nL of protease mix was added (1x NEB CutSmart buffer, 1.21 mg/mL ProteinaseK (Roche, 000000003115836001)) and the plate was incubated at 50 °C for 10 hours and 80 °C for 20 minutes. Next, 230 nL DpnI mix was added (1x NEB CutSmart buffer, 0.2 U NEB DpnI) and the plate was incubated at 37 °C for 4 hours and 80 °C for 20 minutes. Finally, 50 nL of DamID2 adapters were dispensed (final concentrations varied between 32 and 128 nM), together with 450 nL of ligation mix (1x T4 Ligase buffer (Roche, 10799009001), 0.14 U T4 Ligase (Roche, 10799009001)) and the plate was incubated at 16 °C for 12 hours and 65 °C for 10 minutes. Contents of all wells with different primers and adapters was pooled and incubated with 0.8 volume magnetic beads (CleanNA, CPCR-0050) diluted 1:4 or 1:8 with bead binding buffer (20% PEG8000, 2.5M NaCl) for 10 minutes, washed twice with 80% ethanol and resuspended in 7 μL nuclease-free water before in vitro transcription at 37 °C for 14 hours using the MEGAScript T7 kit (Invitrogen, AM1334). Library preparation was done as described in the CEL-Seq protocol with minor adjustments[Hashimshony, T. et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome biology 17, 77, (2016).]. Amplified RNA (aRNA) was cleaned and size-selected by incubating with 0.8 volume magnetic beads (CleanNA) for 10 min, washed twice with 80% ethanol and resuspended in 22 μL nuclease-free water, and fragmented at 94 °C for 2 minutes in 0.2 volume fragmentation buffer (200 mM Tris-acetate pH 8.1, 500 mM KOAc, 150 mM MgOAc). Fragmentation was stopped by addition of 0.1 volume fragmentation STOP buffer (0.5 M EDTA pH8) and quenched on ice. Fragmented aRNA was incubated with 0.8 volume magnetic beads (CleanNA) for 10 minutes, washed twice with 80% ethanol and resuspended in 12 μL nuclease-free water. Library preparation was done as previously described[Hashimshony, T. et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome biology 17, 77, (2016).] using 5 μL of aRNA and PCR cycles varied between 8 and 10. Libraries were run on the Illumina NextSeq platform with high output 75 bp paired-end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3624706