title: GSM3370006: hESC; Homo sapiens; RNA-Seq
library_construction_protocol: Single cells were flow sorted into a chilled 384-well PCR plate containing 1.2μl of primer/lysis mix [20 nM indexed polydT primer, 1:6.000.000 dilution of ERCC RNA spike-in mix (Ambion - 4456740), 1 mM dNTPs, 1.2 units SUPERaseIN Rnase Inhibitor (Thermo Fisher - AM2696), DEPC water (Thermo Fisher - AM9920)] using a BD FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA) and the protocol described above. Sorted plates were sealed, centrifuged for 1 min at 3000 rpm and immediately frozen upside down at -80C until further processing using an adapted CelSeq2 protocol [51]. Cell sorting and preparation: Single cells were flow sorted into a chilled 384-well PCR plate containing 1.2μl of primer/lysis mix [20 nM indexed polydT primer, 1:6.000.000 dilution of ERCC RNA spike-in mix (Ambion - 4456740), 1 mM dNTPs, 1.2 units SUPERaseIN Rnase Inhibitor (Thermo Fisher - AM2696), DEPC water (Thermo Fisher - AM9920)] using a BD FACSAria III flow cytometer (BD Biosciences, San Jose, CA, USA) and the protocol described above. Sorted plates were sealed, centrifuged for 1 min at 3000 rpm and immediately frozen upside down at -80C until further processing using an adapted CelSeq2 protocol [51]. cDNA synthesis: Sorted plates were thawed on ice and briefly centrifuged. To lyse the cells and anneal the mRNA capture primer the plate was incubated at 65C for 5 min and immediately chilled on ice for at least 2 min before adding 0.8μl reverse transcription reaction mix [in 2μl RT reaction: 1x Fist Strand buffer (Invitrogen - 18064-014), 20 mM DTT (Invitrogen - 18064-014), 4 units RNaseOUT (Invitrogen 10777-019), 10 units SuperScript II (Invitrogen 18064-014)]. The plate was incubated at 42oC for 1 hr, 70C for 10 min and chilled to 4C to generate first strand cDNA. For second strand cDNA synthesis 6μl of second strand reaction mix were added [1x NEBNext Second Strand Synthesis buffer (NEB #E6111S), NEBNext Second Strand Synthesis Enzyme Mix: 2.4 units DNA Polymerase I (E. coli), 2 units RNase H, 10 units E. coli DNA Ligase (NEB #E6111S), DEPC water (Thermo Fisher - AM9920)]. The plate was incubated at 16oC for 2 hrs to generate double stranded cDNA. Pooling and In-vitro-transcription: All samples were pooled and cleaned using a 1.2X NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel - 7449970.5) according to manufactures instruction. To reduce the amount of beads for each 100μl pooled sample 20μl beads and 100μl bead binding buffer (20% PEG8000, 2.5M Nacl, pH5.5) was added. The cDNA was eluted in 6.4μl DEPC water and kept with beads for the following IVT reaction were 9.6μl of IVT reaction mix [1.6μl of each of the following: A,G,C,U, 10X T7 buffer, T7 enzyme (MEGAscript T7 Transcription Kit – Ambion AM1334)] was added and incubated at 37C for 13 hrs and then chilled and kept at 4 C. To remove leftover primers 6μl ExoSAP-IT For PCR Product Clean-Up (Affymetrix - 78200) was added and the sample was incubated at 37oC for 15 min and then chilled and kept at 4oC. RNA fragmentation and cleanup: Chemical heat fragmentation was performed by adding 5.5μl of 10X Fragmentation buffer (RNA fragmentation reagents - AM8740) to the sample and incubation in pre-heated thermal cycler at 94C for 2.5 min followed by immediately chill on ice and addition of 2.75μl of Fragmentation Stop buffer (RNA fragmentation reagents - AM8740). The fragmented amplified RNA was purified using 1.8X RNAClean XP beads (Beckman coulter - A63987) according to manufactures instruction and eluted in 6 μl DEPC water of which 5μl (no beads) were transferred to a fresh tube for library preparation. 5'-tagged-random-hexamer reverse transcription (ranhexRT): The fragmented RNA was transcribed into cDNA using 5'-tagged random hexamer primers (GCCTTGGCACCCGAGAATTCCANNNNNN) introducing a partial Illumina adapter as also described in [51]. To remove RNA secondary structure and anneal the mRNA capture primer 1μl of tagged random hexamer (100 μM) and 0.5μl of 10mM dNTPs (dNTP solution set NEB - N0446S) were added to the sample and incubated at 65oC for 5 min and immediately chilled on ice for at least 2 min before adding 4μl reverse transcription reaction mix [in 10μl RT reaction: 1x First Strand buffer (Invitrogen - 18064-014), 20 mM DTT (Invitrogen - 18064-014), 4 units RNaseOUT (Invitrogen 10777-019), 10 units SuperScript II (Invitrogen 18064-014)]. Library amplification and cleanup: The PCR primers introduce the full-length adaptor sequence required for Illumina sequencing (for details see Illumina small RNA PCR primers). PCR was performed in 12.5μl using half of the ranhexRT sample as a template [1X KAPA HiFi HotStart ReadyMix (KapaBiosystems KK2602), 400 nM each primer]. The final PCR amplified Library was submitted to two consecutive 1x NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel - 7449970.5) according to manufactures instruction. The final library was eluted in 20μl of 10 mM Tris-HCl solution (Sigma-Aldrich – T2319-1L).
attributes.tag: GEO Accession
attributes.value: GSM3370006