title: GSM4409086: Cabin1_KO_2cell_4; Mus musculus; RNA-Seq
library_construction_protocol: The RNA-seq libraries were generated from individually cells using Smart-seq2 with minor modification. Briefly, individually cells were lysed and mRNA was captured and amplified by using Smart-seq2 v4 kit (Clontech). After pre-amplification and AMPure XP beads purification, amplified RNAs were quality checked using Agilent High Sensitivity D5000 kit (Agilent Technologies). For ATACseq libraries prepration, oocytes or embryos were lysed in ice-cold lysis buffer (10mM Tris-HCl (pH 7.4), 10mM NaCl, 3mM MgCl2 and NP-40 (0.5%) for 15 minutes on ice to prepare the nuclei. High quality of amplified RNAs were subject to library preparation using Nextera XT DNA Library Preparation Kit (Illumina) and multiplexed by Nextera XT Indexes (Illumina). The quality and quantity of sequencing libraries were determined using Qubit 3.0 assay (Life Technologies) and Agilent High Sensitivity kit at Tapestation 4200 system (Agilent), respectively. For ATAC-seq library preparatio, immediately after lysis, Nuclei were then incubated with the Tn5 transposase (TDE1) and tagmentation buffer at 37°C for 30 minutes with shaking on a thermomixer at 500g. After the tagmentation, 0.5 ul of 10% SDS was added directly into the reaction to end the tagmentation and tagmentated DNA was purified using MinElute Reaction Cleanup Kit (Qiagen). PCR was performed to amplify the ATAC-seq libraries using Illumina TrueSeq primers and multiplex by indexes primers with the following PCR conditions: 72°C for 3min; 98°C for 30s; and 15 thermocycling at 98°C for 15s, 60°C for 30s and 72°C for 3min; following by 72°C 5 min. After the PCR reaction, libraries were purified with the 1.1X AMPure beads (Beckman). All sequencing libraries were pooled and then sequenced on the Illumina HiSeq X platform with 150-bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM4409086