GSM1978027: Tet_TKO_E6.5_Epiblast_1; Mus musculus; RNA-Seq
Total RNA was purified from isolated epiblasts by using the Qiagen AllPrep DNA/RNA Mini Kit, treated with DNase. About 1 ng of extracted RNA was first amplified 18 cycles according to the protocol for single-cell RNA-seq published previously. Briefly, 1 ng of extracted RNA was reverse translated into cDNA, then the free primers were removed by the ExoSAP-IT. TdT was used to add PolyA to the 3’ end of cDNA, the second strand were synthesized, and the double-stranded DNA product were amplified 18 cycles to obtain enough product for DNA library construction. The amplified DNA product was purified by DNA Clean & Concentrator™-5 (Zymo Research), and further purified by Zymoclean™ Gel DNA Recovery Kit (Zymo Research) for the 0.5-8 kb size DNA products. The gel purified products were sheared by Covaris S2 for the generation of fragments about 250 bp in length, and purified with DNA Clean & Concentrator™-5 (Zymo Research). Then NEBNext® UltraTM DNA Library Prep Kit for Illumina® (NEB, Catalog #E7370L) was used to construct a cDNA library, with reagent amount reduced to half of the reactions. The cDNA libraries were sequenced on Illumina HiSeq 2500 instrument with 100-bp reads and paired-end parameter. single cell RNA-seq
GEO Accession
GSM1978027