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==June 2013== ===June 2, 2013=== [[File: 2013_06_02_hela5before.tif|400px]] Cell 5, HeLa, before expelling onto glass top tube cap [[File: 2013_06_02_hela5after.tif|400px]] Cell 5, HeLa, after expelling. The cell here is in the lower left of center. The other two objects are appear small, irregular, and shriveled as the focal plane is moved through them. This is difficult to convey in a single image. [[File: 2013_06_02_hela6before.tif|400px]] Cell 6, HeLa, before expelling [[File: 2013_06_02_hela6after.tif|400px]] Cell 6, HeLa, after expelling. There legitimately appears to multiple cells in this droplet. * These samples were diluted to ~10,000 cells/ml --> 10 cells/ul. More dilution might help avoid picking multiple cells, but other technical challenges remain. * After depositing the cell on the glass top tube cap, there is only ~10 seconds before the droplet dries (~100 nl maximum volume by my estimate, although variance is large.) This is hardly enough time to image the cell. * The CEL-Seq paper refers to "pipetting off excess liquid." I haven't tried this yet. I could deposit the cell in a larger volume, and then adjust the volume down. This issue here would be finding the cell in that larger volume (if I fail to see it shoot out of the pipette.) Also, the omnipresent dust particle and cell debris make imaging the cells very hard in BF. * I should probably wash the cells after thawing. ===June 25, 2013=== * Implemented Quartz seq, Samples A1 and A2. ** Original Quartz-seq protocols: http://bit.accc.riken.jp/protocols/ ** Sample A1: 500 pg UHRR, CEL-Seq equivalent ERCC, Quartz purified RNA protocol. Used 2x Terra Direct PCR Mix. ** Sample A2: Same, except I used 2x KAPA HiFi Uracil+. * Both samples had 1 ul of 1:500 dilution of 100 mM dUTP during final PCR (107 ul total volume). ====Quartz-Seq Protocol for 500 pg Agilent UHRR, 2 samples==== * Work in the PCR hood * Keep enzyme stocks in cold box * Mix reactions on cold rack * Use UV treated Ambion RNase/DNase free water * Use UV treated 0.5 and 1.5 ml Eppendorf Lo-Bind tubes * Use UV treated 0.2 ml Axygen Cat# 22-154LR low retention tubes Sequences used in Quartz protocol: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Name''' | align="center" style="background:#f0f0f0;"|'''Sequence''' |- | RT primer||TATAGAATTCGCGGCCGCTCGCGATAATACGACTCACTATAGGGCGTTTTTTTTTTTTTTTTTTTTTTTT |- | Tagging primer||TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT |- | Suppression PCR primer||GTATAGAATTCGCGGCCGCTCGCGAT |- | |} * UHRR/ERCC mix: 2.5 ul 500 pg/ul UHRR 2.5 ul 1:1e4 ERCC 14 ul H2O -------------- Total = 19 ul * RT annealing mix: 3 ul 10X Titanium Taq buffer 3 ul 1 mM dNTPs 3 ul 0.833 uM RT primer 2 ul RNasin Plus 19 ul UHRR/ERCC mix ---------------------------- Total = 30 ul * RT rxn mix: 2 ul 10X Titanium Taq buffer 13 ul H2O 2.5 ul 0.1 M DTT 2.5 ul SuperScript III ----------------------------- Total = 20 ul * RT annealing rxn: 12 ul RT priming buffer (Quartz seq adds this to beads, but we are using purified reference RNA in the priming buffer) --------------------------------------------------------------------------------------------------- Total = 12 ul # 1 min @ RT # 90 sec @ 70 degC # 15 sec @ 35 degC # Hold @ 4 degC * RT rxn: 12 ul RT annealing rxn 8 ul RT rxn mix ---------------------- Total = 20 ul # 5 min @ 35 degC # 20 min @ 45 degC # 10 min @ 70 degC # Hold at 4 degC # Prepare Exo I mix * Exo I mix: 2 ul 10X Titanium Taq buffer 1 ul 10X Exo III buffer 1 ul 0.1 M DTT 3 ul Exo I 23 ul H2O ----------------------------- Total = 30 ul * RT primer removal: ** 20 ul RT rxn ** 36 ul Ampure RNAClean XP ** Total = 56 ul # 10 min @ RT # 5 min on magnet # Wash 2x with 1 min with 50 ul 80% EtOH # Dry 3 min # Add 6 ul Exo I mix # 1 min @ RT # 5 min on magnet # Transfer to new tube # 30 min @ 37 degC # 20 min @ 80 degC # Hold @ 4 degC * PolyA mix: 1 ul 10X Titanium Taq buffer 1.5 ul 20 mM dATP (dilute 100 mM dATP 1:5) 1.2 ul 1:5 RNase H 0.84 ul TdT (Roche) 5.46 ul H2O ------------------------------------------ Total = 10 ul * PolyA rxn: 6 ul Exo I rxn 5 ul PolyA mix -------------- Total = 11 ul # 50 sec @ 37 degC # 10 min @ 65 degC # Hold @ 4 degC * 2nd strand mix: 78.25 ul 2x Terra buffer 1 ul 10 uM Tagging primer 6.25 ul Terra polymerase 58.75 ul H2O ------------------------- Total = 144.25 ul * 2nd strand rxn: 11 ul PolyA rxn 46 ul 2nd strand mix -------------------- Total = 57 ul # 10 sec @ 98 degC # 1 min @ 40 degC # 5 min @ 68 degC # Hold @ 4 degC # Move to cold rack ---------------------------------- * PCR mix (Clontech): 21.1 ul 2x Terra 1 ul 100 uM SuppressPCR primer 25.25 ul H2O ------------------------------ Total = 52.33 ul * PCR mix (KAPA): 21.1 ul 2x HiFi U+ 1 ul 100 uM SuppressPCR primer 25.25 ul H2O ------------------------------ Total = 52.33 ul * PCR rxn: 57 ul 2nd strand rxn 50 ul PCR mix --------------------- Total = 107 ul * Clontech protocol: # Preheat, 10 sec @ 68 deg # 15 cycles: ## 10 sec @ 98 degC ## 15 sec @ 65 degC ## 5 min @ 68 degC # 5 min @ 68 degC # Hold @ 4 degC * KAPA protocol # 5 min @ 95 degC # 15 cycles: ## 20 sec @ 98 degC ## 15 sec @ 65 degC ## 5 min @ 72 degC # 5 min @ 72 degC # Hold @ 4 degC ---------------------------------- * Store @ -80 degC until library prep ===June 26, 2013=== * Library prep for Quartz-seq samples A1 and A2: ** Adapter ligation using KAPA Rapid Ligation, USER, and PCR with KAPA SYBR Fast (See Jeff and Noi's protocols). I used NEBNext Index Primers (the loop adapters). The following is directly from the NEBNext Multiplex Oligos for Illumina manual. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Product''' | align="center" style="background:#f0f0f0;"|'''Index Primer Sequence''' | align="center" style="background:#f0f0f0;"|'''Expected Index Primer Sequence Read''' |- | NEBNext Index 11 Primer for Illumina||5Β΄-CAAGCAGAAGACGGCATACGAGAT[GTAGCC]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3Β΄||GGCTAC |- | NEBNext Index 12 Primer for Illumina||5Β΄-CAAGCAGAAGACGGCATACGAGAT[TACAAG]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3Β΄||CTTGTA |- | |} I used Index 12 for sample A1 (Terra) and Index 11 for Sample A2 (KAPA HiFi Uracil+). ====CORE fragmentation and library prep==== * Work in PCR hood * UV treat H2O, tips, tubes * Purified Quartz WTA products using Ampure XP beads ** Added 100 ul beads to 107 ul product ** Eluted in 14.2 ul H2O * UDG/Endo IV rxn 14.2 ul template 1 ul 1:121 UDG 1 ul 1:121 Endo IV 1.8 ul Exo- buffer ------------------ Total = 18 ul # 2 hrs @ 37 degC # 15 min @ 65 degC # Hold @ 4 degc ----------------------------------- * Nick translation 18 ul UDG/Endo IV rxn 1 ul 1:50 Exo- 1 ul 1:25 1 mM dNTP (40 uM) --------------------------- Total = 20 ul # 1 hrs @ 37 degC # 15 min @ 75 degC # Hold @ 4 degC ----------------------------------- * Ligation rxn 20 ul nick translation rxn 25 ul 2X KAPA ligation buffer 1.67 ul 1.5 uM loop adapter 1.33 ul H2O KAPA DNA ligase ----------------------------- Total = 50 ul # 15 min @ 20 degC # Add 2 ul USER # 15 min @ 37 degC # Hold @ 4 degC ----------------------------------- * Ampure XP bead purification ** 50 ul beads ** Elute in 20 ul H2O * PCR 20 ul template 1 ul 10 uM PCR_F 1 ul 10 uM NEBNext index 3 ul H2O 2X KAPA SYBR mix ------------------------ Total = 50 ul # 30 sec @ 98 degC # 15 cycles: ## 10 sec @ 98 degC ## 30 sec @ 65 degC ## 45 sec @ 72 degC # 2 min @ 72 degC # Hold @ 4 degC ------------------------------------- [[File: 2013_06_26 quartz seq 500 pg uhrr.jpd|400px]] I size selected the KAPA library (Index 11) and gave it to Alan for sequencing ([[Arichard:Samples/Sequencing_samples|AR_QC_500pgUhrrKapa_Jun26]]). ===June 27, 2013===
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