Name: GSM7980610
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Single embryo was flushed out from the mouse oviduct, and immediately lysed in 1ul lysis solution containing 1% NP-40 and 4U RNase inhibitor A combination of 1ul of 5x 1st strand buffer and 0.1 ul of RNA spike-in or water was added into the single embryo lysis, followed by the incubation at 82C for 12 minutes. After quick chill on ice, the RNAs were next end repaired by adding 1ul PNK solution (70mM tris-HCl pH7.6, 10mM MgCl2, 15mM DTT, 4 units/ul RNase Inhibitor) containing 2 units of T4 PNK and by incubating at 37C for 10 minutes. The RNA samples were further incubated at 37C for 5 minutes after the addition of 0.5ul mix containing 1 unit of PolyA polymerase and 3 picomole ATP. Before reverse transcription, 1ul of pre-RT mix (5uM R-dT Primer (TTTCCCTACACGACGCTCTTCCGATCTNNNNNNTTTTTTTTTTTTTTTVVN), 6.25mM dNTPs, 15mM EDTA) was added to samples, followed by the heat up at 72C for 2 minutes. After RNA denaturation, samples were immediately chilled on ice, followed by the addition of 5.5ul freshly made RT mix containing 1X 1st strand Buffer, 10mM DTT, 2U/ul RNase inhibitor, 2M Betaine, 1uM GGrG oligo (iCiGiCGGAGTTCAGACGTGTGCTCTTCCGATCTrGrG+G) and 20U/ul Superscript II reverse transcriptase II. Samples were then incubated with the following temperature setting: 40C for 5 mins, 42C for 30 mins, Two cycles of 50C for 2 mins followed by 42C for 2mins, and 15 mins at 70C. First strand cDNA libraries were next amplified by 15 cycles of first round PCR (F: GTGACTGGAGTTCAGACGTGTGCTCTTC; R: ACACTCTTTCCCTACACGACGCTCTTC) in a total volume of 30ul PCR reactions and purified with Ampure XP beads at 1:1 ratio. Purified cDNAs from the PCR (13.5ul) were mixed with 130ng of RNA probes in total 30ul of hybridization buffer (10mM Tris-HCl, pH8.0, 0.5M NaCl, 5mM EDTA and 0.2% SDS), and incubated at the following temperatures: 94C for 2 mins, followed by ramp down to 50C with 1C down per 50 seconds, and finally at 50C for 5 mins. Meanwhile, 100ul of streptavidin C1 beads were washed, pre-blocked and warmed to 50C. To delete ribosomal cDNAs, samples and beads were mixed at 50 C and incubated for another 5 mins at 50 C, and immediately put on a magnetic rack. Supernatant was collected and purified with Ampure XP beads. The purified cDNAs were further amplified by 9 cycles of PCR with Multiplex oligos (NEB). The final PCR product was purified and subject to sequencing.